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Chemico-Biological Interactions 166 (2007) 163–169
Atmospheric photochemical transformations enhance1,3-butadiene-induced inflammatory responses in
human epithelial cells: The role of ozone and otherphotochemical degradation products�
Melanie Doyle a,∗, Kenneth G. Sexton a, Harvey Jeffries a, Ilona Jaspers b
a Department of Environmental Sciences and Engineering, University of North Carolina-Chapel Hill,CB #7431, Chapel Hill, NC 27599, USA
b CEMLAB, University of North Carolina, Chapel Hill, NC, USA
Available online 3 June 2006
bstract
Chemistry of hazardous air pollutants has been studied for many years, yet little is known about how these chemicals, once reactedithin urban atmospheres, affect healthy and susceptible individuals. Once released into the atmosphere, 1,3-butadiene (BD) reactsith hydroxyl radicals and ozone (created by photochemical processes), to produce many identified and unidentified products. Once
his transformation has occurred, the toxic potential of atmospheric pollutants such as BD in the ambient environment is currentlynclear. During this study, environmental irradiation chambers (also called smog chambers), utilizing natural sunlight, were used toreate photochemical transformations of BD. The smog chamber/in vitro exposure system was designed to investigate the toxicity ofhemicals before and after photochemical reactions and to investigate interactions with the urban atmosphere using representativen vitro samples.
In this study, we determined the relative toxicity and inflammatory gene expression induced by coupling smog chamber atmo-pheres with an in vitro system to expose human respiratory epithelial cells to BD, BDs photochemical degradation products, orhe equivalent ozone generated within the photochemical mixture. Exposure to the photochemically generated products of BD
primarily acrolein, acetaldehyde, formaldehyde, furan and ozone) induced significant increases in cytotoxicity, IL-8, and IL-6 genexpression compared to a synthetic mixture of primary products that was created by injecting the correct concentrations of theetected products from the irradiation experiments. Interestingly, exposure to the equivalent levels of ozone generated during thehotochemical transformation of BD did not induce the same level of inflammatory cytokine release for either exposure protocol, uggesting that the effects from ozone alone do not account for the entire response in the irradiation experiments. These resultsndicate that BDs full photochemical product generation and interactions, rather than ozone alone, must be carefully evaluated whennvestigating the possible adverse health effects to BD exposures. The research presented here takes into account that photochemical� This publication has not been formally reviewed by the American Chemistry Council. The views expressed in this document are solely thosef Melanie Doyle, Dr. Kenneth Sexton, Dr. Harvey Jeffries, and Dr. Ilona Jaspers. Although the research described in this article has been funded whollyr in part by the United States Environmental Protection Agency through cooperative agreements CR829522 with the Center for Environmentaledicine, Asthma, and Lung Biology and R829762 with the Department of Environmental Science and Engineering at the University of Northarolina at Chapel Hill, it has not been subjected to the Agency’s required peer and policy review, and therefore does not necessarily reflect the views of
he Agency and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement orecommendation for use.∗ Corresponding author. Tel.: +1 919 966 1372; fax: +1 919 966 7911.
E-mail address: [email protected] (M. Doyle).
009-2797/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.cbi.2006.05.016
164 M. Doyle et al. / Chemico-Biological Interactions 166 (2007) 163–169
transformations of hazardous air pollutants (HAPs) does generate a dynamic exposure system and therefore provides a more realisticapproach to estimate the toxicity of ambient air pollutants once they are released into the atmosphere.© 2006 Elsevier Ireland Ltd. All rights reserved.
spheric
Keywords: Air pollution; 1,3-Butadiene; Irradiation chambers; AtmoPhotochemical products; Secondary air toxics1. Introduction
BD is not a new topic of concern when studyinghuman health and the atmosphere. The Clean Air ActAmendments of 1990 added BD to EPAs hazardous airpollutants list, it ranks 36th in the top 50 most producedchemicals within the United States [1], and is one of thetop 33 in the Toxic Release Inventory [2]. With fluctu-ating emissions from both biogenic and anthropogenicsources [3–5] reaching as high as 3000 tonnes per yearwithin the US [6], the fate and transport of BD in theatmosphere plays an important role in the overall healthconcern after examining releases into the environment.
Once released into the atmosphere, BD reacts throughpartially known chemical mechanisms, such as reactionswith hydroxyl and other radicals as well as ozone, whichare all created during photochemical processes. Duringthe daytime, considering only BD reacting with hydroxylradicals (with average OH concentrations of approxi-mately 2 × 106 molecule/cm3), BD has a lifetime in theatmosphere of only 1–2 h [7], supporting the significancefor studying the adverse effects of the generated transfor-mation products. These atmospheric reactions result inthe formation of products, some that have been identifiedand have quantified yields, some that have been identi-fied only, some that have been detected but not identified,and some products that have yet to be detected [7–11].Because of the latter two conditions, studying BDs fulltoxic potential is difficult: an investigator would not evenknow (nor would likely have available) all the degrada-tion products to consider or test.
Known BD photochemical degradation productsinclude acrolein, formaldehyde, organic nitrates, 1,2-epoxy-3-butene (butadiene monoxide), CO, CO2,ozone, PAN, furan, glycolaldehyde, glycidaldehyde,3-hydroxy-propanaldehyde, malonaldehyde [7,12–17].Many of these products, i.e. ozone and formaldehyde,are not merely formed through reactions with BD, butare generated during other photochemical transforma-
tion processes of most other volatile organic compounds(VOC) that are readily available in the ambient atmo-spheric environment.Although many studies have shown the adverseeffects of BD and its known photochemical transfor-
chemistry; In vitro; Lung epithelial cells; Interleukin 8; Interleukin 6;
mation products, considered separately, little literaturecould be found that studied realistic ambient concentra-tions, non-carcinogenic end-points, or short-term expo-sures. The UNC smog chambers have been used for morethan 30 years to investigate and develop chemical mech-anisms of atmospheric species including all of those usedin regulatory air quality models. Recently, these cham-bers have been combined with an in vitro cell exposuresystem to better facilitate air quality research. This sys-tem was created to allow the study of effects occurring inhuman respiratory epithelial cells, using complete pho-tochemical transformation mixtures naturally generatedin ambient environmental conditions and without alter-ing the natural chemical mixture state. BD and all of itsknown primary or first generation photochemical prod-ucts (primarily acrolein, formaldehyde, acetaldehyde,furan, ozone) are known respiratory irritants [18–27], butno literature could be found on what respiratory effectsmight occur with these irritants when an individual issimultaneously exposed to them all, for example in aneighborhood downwind of a polymer facility after anunplanned release of BD from their process system.
One large concern when working with pollutant mix-tures and testing their toxicity on human respiratoryepithelial cells is how to identify which toxic agent iscausing the adverse effects. This paper illustrates onemethod to attack this problem. Here we examine thecytotoxicity and inflammatory gene expression inducedfrom exposure (1) to the whole BD reaction systemproduct set; (2) to ozone alone, but at concentrationsequal to those generated during photochemical reactionsof BD complex mixtures; (3) to synthetic mixtures ofthe detected and quantified products of BD observed inthe full photochemical system mixture. We can there-fore assess the importance of the BD products and moreimportantly, assess the importance of the detected-notqualified and the undetected but present products. Thisstudy is an extension of previous work examining theadverse effects of BD on human respiratory epithelialcells [28]. Those results indicated that photochemical
transformations significantly alter the toxicity of BDmixtures when examining cytotoxicity and IL-8 geneexpression only. Taken together with other publishedfindings, this study was conducted to evaluate the toxic
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otential of BDs photochemical transformation prod-cts, and to possibly decipher which photochemicalransformation product, if any, significantly contributedo the overall response found from the full photochemi-ally generated product mixture.
. Materials and methods
.1. Smog chambers
Outdoor environmental irradiation chambers (alsoalled smog chambers) that utilize natural sunlight cane used to study systems of natural transformation chem-stry of pollutants [28–31]. Dual 150,000-l UNC outdoormog chambers made of FEP Teflon film were useds photochemical reactors for these experiments. Thehambers are located in Chatham County, NC. Descrip-ive information about the chambers has been published29,30]. These chambers are ideal to study chemical sys-ems that are part of the real photochemical phenomenaccurring within the ambient atmosphere because theEP Teflon film allows the transmission of the importantltraviolet and visible regions of sunlight that initiate theransformation processes.
Two injection protocols were used to discriminatehe effects induced by different products generated dur-ng photochemically active systems representative ofD released into the environment. The chemical sys-
em requires oxides of nitrogen (NO and NO2) as wells the VOC to react. On one side of the chamber, wenjected 200 ppbV butadiene (National Specialty Gases,urham, NC) and 50 ppb NO in the early afternoon and
hese were allowed to react with sunlight until sundownapproximately 5 h). After sundown, one of two differentystems were created in the second chamber: (1) a syn-hetic blend of BDs first generation products (acrolein,zone, and formaldehyde) equal to the concentrationsetected in the other chamber or (2) the amount of ozonelone that was created during BDs reaction. Using thesewo protocols, we can evaluate not only the effects due tohe ozone created in the photochemically active system,ut also the combined effects due to a majority of thenown, first generation products.
.2. Cell culture and in vitro exposure
A549 cells, a human pulmonary type II epithelial-likeell line, were cultured in F12K medium plus 10% fetal
ovine serum and 1% penicillin and streptomycin (allrom Invitrogen, Carlsbad, CA) [28,31,32]. Upon con-uency, the culture medium was replaced with serum-ree media (F12K, 1.5 �g/ml bovine serum albumin, andteractions 166 (2007) 163–169 165
antibiotics). Just before transport to the smog chambersite, media located in the apical chamber was aspirated,while media in the basolateral compartment remained.This facilitates direct exposure of lung epithelial cellsto gaseous pollutants without significant interference ofmedia, yet the cells are maintained with nutrients fromthe basolateral side. Different sets of cells were exposedfor 5 h to smog chamber mixtures comprised of (1) thephotochemically generated products of BD, (2) a syn-thetic blend of BDs first generation products (199 ppbacrolein, 171 ppb ozone, and 87 ppb formaldehyde), or(3) alternatively, the amount of ozone created in the fullmixture.
2.3. Smog chamber–lung cell exposure system
A schematic of the smog chamber–lung cell expo-sure system was previously published [28,31]. Outdoorenvironmental chambers were interfaced with modu-lar incubator chambers composed of 8-liter cell expo-sure units. Sample lines, directly coupled to the smogchambers through two externally circulated sample man-ifolds, were used to provide chamber gases to the cellsduring exposure [31]. Three cell exposure units wereused throughout these studies, two of which were sup-plied with chamber air from the gas mixtures generatedwithin the dual smog chamber and one was supplied withhumidified medical-grade clean air. Both cell exposuresystems were mixed with 5% CO2. For each experiment,another set of A549 cells was exposed to clean air to con-trol for potential variations induced by tissue culture ortransport of the cells. In addition, the clean air controlcell exposure chamber was used to hold the cells duringpre- and post-exposure periods.
2.4. Chemical analysis
During each experiment five gas chromatographic(GC) methods were used to monitor volatile organiccompounds within the chambers. One GC (Carle Inc.,Chandler Engineering, Tulsa, OK) was used to measuretotal hydrocarbon (THC), which was used for assuringlow background concentrations while also measuringthe initial injections. Samples were taken continuallythroughout the experiment, once an hour from eachchamber and analyzed with two GCs (Carle Inc.) usingpacked isothermal columns coupled to flame ionizationdetectors (FID). A Varian 3700 GC with electron cap-
ture detector (ECD), used to measure CCl4 (our dilutiontracer), PAN, and other N- or O-containing compounds,was also used continually every 30 min throughout theduration of the experiment. A Varian 3400 capillary GC-
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166 M. Doyle et al. / Chemico-BioloFID was used with a Varian Saturn 2000 ion trap massspectrometer to analyze air samples taken before, at thebeginning, and during each exposure to help analyze forboth known and unknown products created during thephotochemical reactions. Formaldehyde was measuredcontinuously, using the automated Dasgupta-diffusion-tube sampler to obtain aqueous formaldehyde, whichis then mixed with buffered 2,4-pentanedione and mea-sured with fluorescence [33]. Ozone was measured usingan EPA standard reference method (EQOA-0880-047)based on photometry with a Thermo EnvironmentalInstruments Inc., Model 49 monitor. Nitrogen oxideswere measured using an EPA standard reference method(RFNA-1292-090) based on chemiluminescence with aMonitor Labs Incorporated Model 98-41 monitor. BothEPA reference methods are described in Title 40 Codeof Federal Register Part 53 “Ambient Air MonitoringReference and Equivalent Methods”.
2.5. Analysis of cytotoxicity and cytokines
Approximately 9 h post-exposure basolateral super-natants from the exposed cells were stored at −80 ◦Cuntil analysis for cytotoxicity and inflammatory geneexpression. For the analysis of cytotoxicity, the baso-lateral supernatants were analyzed for the release of celllactate dehydrogenase (LDH) using a coupled enzymaticassay (Takara-Bio, Japan), as per the suppliers instruc-tions. Cytotoxicity was expressed as LDH levels withfold increase over the individual clean air control.
Basolateral supernatants were analyzed for cytokineprotein levels by ELISA (R&D Systems, Minneapolis,MN or Biosource, Camerillo, CA), as per the supplier’sinstructions. Protein levels were adjusted to account forthe differences in viable cells that could produce andrelease cytokines into the supernatant and expressed asfold increase over the individual clean air control.
2.6. Statistical analyses
The results were compared and analyzed using twostatistical methods, one-way and two-way analysis-of-
Table 1Chemical analysis of chamber constituents chamber concentrations (ppmC)
BD Acrolein
Ave. Max. Ave. M
BD + NOx + light 0.072 0.056 0.190 0Syn. Pro. mixture 0.00 0.00 0.180 0Ozone 0.00 0.00 0.00 0
Ave. is the average concentration the cells were exposed to over the 5-h pexperiment. Syn. Pro. mixture is the synthetic butadiene product mixture.
teractions 166 (2007) 163–169
variance. A one-way ANOVA was used to compare theresponse averages individually against one another usingDunn’s multiple comparison test. A two-way ANOVAwas used to quantify the separate effects induced by thedifferent components of the photochemically generatedmixture using Tukey’s comparison method.
3. Results
In this study human respiratory epithelial cells wereexposed to the primary degradation products formedduring photochemical transformations of BD and nitricoxides in natural systems and examined for cytotoxicityand cytokine gene expression.
The products generated by photochemical transfor-mations of BD and nitric oxides were identified and con-firmed using GC/MS. Table 1 summarizes the averageand maximum concentrations produced for the knownand quantifiable photochemical products derived fromBD during the cellular exposure period. Although amajority of the products were quantified and used tocreate the synthetic product mixture (acrolein, formalde-hyde and ozone), other known products were found atsignificantly smaller concentrations. During each of theexperiments, the cells were exposed to practically thesame concentrations of transformation products (within±0.010 ppm). Cytotoxicity or cell viability was deter-mined through calculation of LDH release induced byexposure to the photochemical degradation products ofBD, or ozone. Fig. 1 displays the LDH response inducedby BDs generated photochemical product mixture, thesynthetic blend of BD products, or ozone alone. BDsprimary photochemical products (a synthetic mixture ofacrolein, formaldehyde and ozone) account for a statis-tically significant portion of the LDH response observedfrom the full product mixture generated within thesmog chambers, compared to the response of the ozone
alone.To examine the proinflammatory potential of BDstransformation products, we compared the effects of BDsgenerated photochemical products, a synthetic mixture
Form Ozone
ax. Ave. Max. Ave. Max.
.202 0.083 0.093 0.146 0.177
.198 0.079 0.087 0.155 0.185
.00 0.00 0.00 0.160 0.182
eriod and Max. is the maximum concentration produced during the
M. Doyle et al. / Chemico-Biological Interactions 166 (2007) 163–169 167
Fig. 1. Cytotoxicity, indicated by increased LDH release, was exam-ined using chamber mixtures. The mixtures compared in the studyinclude: (1) the photochemically generated products of BD, (2) a syn-thetic blend of BDs first generation products (acrolein, ozone, andformaldehyde), and (3) the amount of ozone in the synthetic mixture.T#
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Fig. 3. Analysis of IL-6, indicated by increase in protein release, wasexamined for BD chamber mixtures. The mixtures compared in thestudy include: (1) the photochemically generated products of BD, (2)a synthetic blend of BDs first generation products (acrolein, ozone, andformaldehyde), and (3) the amount of ozone in the synthetic mixture.
he results were expressed as fold increase over the control ± S.E.M.Statistically significant compared to ozone p ≤ 0.05. *Statistically sig-ificant compared to synthetic BD product mixture.
f measured primary products (acrolein, formaldehydend ozone), or the amount of ozone generated during thehotochemical processes using A549 cells. Figs. 2 and 3how the IL-8 and IL-6 response induced by exposureso product mixtures using the smog chamber–in vitroystem. For both mediator responses, the data indicatesn increasing step-wise response from ozone, to ozonend the primary product mixture, to the full generatedhotochemical products. Unlike cytotoxicity, neither the
zone nor the mixture of primary products significantlyccount for a portion of the IL-8 or IL-6 inflammatoryesponse found.ig. 2. Analysis of IL-8, indicated by increase in protein release, wasxamined for BD chamber mixtures. The mixtures compared in thetudy include: (1) the photochemically generated products of BD, (2)synthetic blend of BDs first generation products (acrolein, ozone, and
ormaldehyde), and (3) the amount of ozone in the synthetic mixture.he results were expressed as fold increase over the control ± S.E.M.Statistically significant compared to ozone p ≤ 0.05. *Statistically sig-ificant compared to synthetic BD product mixture.
The results were expressed as fold increase over the control ± S.E.M.#Statistically significant compared to ozone p ≤ 0.05. *Statistically sig-nificant compared to synthetic BD product mixture.
4. Discussion
Previous work using smog chambers to study environ-mental toxicology have demonstrated the importance ofexamining the photochemical transformations of pollu-tants when analyzing their toxic potential once releasedinto the atmosphere [28,31]. While research has beendone on the individual products formed and their adverserespiratory effects [19–27], almost no work could befound that examined how combined exposures to thesepollutants may alter their overall toxicity either throughsynergism or antagonism. Our system which uses out-door irradiation chambers combined with an in vitroexposure unit, provides an improved method to studyphotochemically active pollutants in a holistic setting.This approach enables all of the photochemical transfor-mation products, including the undetected and detectedbut unknown products, to be generated in proper relativeratio to one another. In this study, we examined cyto-toxicity and the release of proinflammatory mediatorsinduced by BD/NOx photochemical gaseous mixturesusing A549 cells, a human alveolar type II-like cell line.Indicators used as markers of inflammation were IL-6and IL-8.
This unique system was used to discriminate betweenthe responses induced by the majority of individualproducts formed during BDs photochemical transfor-mations in the atmosphere. Although many productsare generated during these reactions (and are previously
mentioned), the synthetic product mixture representa-tive of the known, first generation products includedonly acrolein, formaldehyde and ozone. These productsaccount for a large portion of those initially generated
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168 M. Doyle et al. / Chemico-Bioloand were measured with high certainty using GC andGC/MS techniques. The limitations of the hydrocarbonmeasuring techniques used in this study did not allow forthe quantification of other known products such as buta-diene monoxide, glycolaldehyde, and glycidaldehyde;however, they were generated in small quantities andcould be detected.
During the study, cells were exposed to BD/NOx
product concentrations similar to those that could befound in the ambient environment. All three exposureprotocols (BDs generated photochemical products, thesynthetic mixture of BD first generation products, andthe amount of ozone generated during the photochem-ical system) induced significant increases in LDH, IL-8 and IL-6, compared to the clean air control. Whileacute exposures to the combined mixture of BDs photo-chemical products caused significant induction of proin-flammatory mediators, cell viability may be driven pri-marily by the concentrations of ozone generated withineach system. Other products formed through BD trans-formations with known toxicities, i.e. furan, butadi-ene monoxide and PAN, have been measured duringprevious experiments using the outdoor environmen-tal chambers. Although previous studies have shownthese products to cause adverse effects at higher concen-trations, they were not added to the synthetic mixturebecause of the small amounts generated using the cur-rent study’s experimental protocol. Thus, other knownproducts generated from photochemical transformationsof BD need to be evaluated for possible interactionswhen combined with those evaluated here. More impor-tantly is the significant difference in the inflammatoryresponse induced by the full photochemically generatedBD products and that of the synthetic blend or mixtureof primary products. This suggests that both detectedbut unknown and detected but not quantified products,even those formed in small quantities during the photo-chemical transformations of BD, play an important rolein the induction of proinflammatory mediators in A549cells.
Currently we are unable to quantify the individualtoxicities of the unknown or unspecified products, there-fore demonstrating the importance of additional studiesto create new chemical mechanisms for photochemi-cal transformation of hazardous pollutants once releasedinto the atmosphere. Developing these mechanisms toidentify new products could be used to discern possibleinteractions between the photochemical transformation
products generated from BD. In addition, more con-trolled exposure studies are necessary to determine thepotential mechanisms by which exposure to BD photo-chemical transformation products enhance toxicity.teractions 166 (2007) 163–169
Taken together, the data presented here demonstratethat exposure to BDs photochemical transformationproducts induce acute inflammatory responses in humanrespiratory epithelial cells. Although the amount ofozone generated from these conditions contributes to aportion of the given response indicators, it does not, how-ever, account for the IL-8 and IL-6 response. Thus, eventhough ozone concentrations may be a good indicatorof the adverse health potential of photochemical smog,we must examine the entire photochemical mixtures thatare produced, each of the products and their subsequentreactions, to estimate the toxicity on the exposed popu-lation.
While the mixtures caused an acute inflamma-tory response that could be responsible for poten-tial adverse respiratory effects, other data evaluatingcytokine expression in the A549 cells observed dur-ing these experiments indicates that these protectiveresponses do not cause irreversible, cellular damagetherefore repair, differentiation, and proliferation postinflammation is expected (data not shown). To the bestof our knowledge, this technique is the only systemthat enables the evaluation of the adverse effects ofphotochemically reactive HAPs under controlled con-ditions while studying both the known and unknownproducts. This study indicates that use of outdoorenvironmental chambers is an essential and valuabletool when examining the toxicity of atmospheric mix-tures representative of urban environments. Overall,the unique net benefit of this research approach isthe integrated results from a single source of exper-iments. They include time-series concentration mea-surements for many pollutants generated during smogchamber experiments and the toxicological results fromthe in vitro exposures. Combined, these can be used tofurther develop air quality simulation models neededto predict the transformation products necessary forexposure analyses and relative risk assessment calcu-lations.
Acknowledgements
This work was funded by grants from the Environ-mental Protection Agency (R829762 and CR829522)and the American Chemistry Council (#2324).
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