toxicologic methods: controlled human exposures

Toxicologic Methods: Controlled Human ExposuresMark J. UtelIl and Mark W. Frampton2'Department of Environmental Medicine; 2Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester,New York, USA

The assessment of risk from exposure to environmental air pollutants is complex, and involves thedisciplines of epidemiology, animal toxicology, and human inhalation studies. Controlled, quantitativestudies of exposed humans help determine health-related effects that result from breathing theatmosphere. The major unique feature of the clinical study is the ability to select, control, andquantify pollutant exposures of subjects of known clinical status, and determine their effects underideal experimental conditions. The choice of outcomes to be assessed in human clinical studies canbe guided by both scientific and practical considerations, but the diversity of human responses andresponsiveness must be considered. Subjects considered to be among the most susceptible includethose with asthma, chronic obstructive lung disease, and cardiovascular disease. New experimentalapproaches include exposures to concentrated ambient air particles, diesel engine exhaust,combustion products from smoking machines, and experimental model particles. Futureinvestigations of the health effects of air pollution will benefit from collaborative efforts among thedisciplines of epidemiology, animal toxicology, and human clinical studies. Key words: air pollution,asthma, cardiac, chronic obstructive pulmonary disease, experimental design, exposure, gases,human, inflammation, methods, particles, pulmonary function, respiratory. - Environ HealthPerspect 1 08(suppl 4):605-613 (2000).http.//ehpnet 1.niehs. nih.gov/docs/2000/suppl-4/605-613utell/abstract. html

The assessment of risk for acute and/orchronic inhalation of low-level environmen-tal air pollutants is complex. Typically, thedatabase for risk assessment arises from threeseparate investigational approaches: epidemi-ology, animal toxicology, and human inhala-tion studies. Carefully controlled quantitativestudies of exposed humans utilize laboratoryatmospheric conditions, considered relevantto outdoor pollutant levels, or concentratedparticles from the ambient air, and documenthealth-related effects that result from breath-ing the atmosphere. Advantage is taken of thehighly controlled environment to identifyresponses to individual pollutants and charac-terize exposure-response relationships. Inaddition, the controlled environment pro-vides an opportunity to study interactionamong pollutants per se or with other vari-ables such as exercise, humidity, or tempera-ture. Insofar as individuals with acute andchronic cardiopulmonary diseases can partic-ipate in exposure protocols, potentially sus-ceptible populations can be studied.However, controlled human exposures haveimportant limitations: for practical and ethi-cal reasons, studies are limited to smallgroups, presumably representative of largerpopulations, to short durations of exposure,and to pollutant concentrations expected toproduce only mild and transient responses.Furthermore, efforts to predict chronichealth effects from acute transient responsesin clinical studies lack validation.

In this article we examine the exposuremethodologies and study design issues essentialfor performance of controlled clinical studies.

Subsequently, we focus on the investigativetools available to evaluate respiratory, cardio-vascular, and pharmacokinetic outcomes.Finally, our current understanding ofresponses of susceptible populations as deter-mined by the clinical study is reviewed.

Experimental Designand MethodsControlled human exposure studies of inhaledgases and particles, especially those designedto evaluate short-term effects of airborne pol-lutants, have been performed in multiple labo-ratories over the past decade. The majorunique feature of the dinical study is the abil-ity to select, control, and quantify pollutantexposures of subjects of known clinical statusand determine their effects on cardiopul-monary performance under ideal conditionsfor conducting physiologic evaluations.

Typically, modern clinical studies useexposures to single pollutants or simple pollu-tant mixtures, exposure durations of0.5-8 hr, and a double-blind, crossoverdesign. Exposures are often conducted inenvironmentally controlled chambers (25-75m3 volume) with a single passage of the pol-lutant(s). Physiologic responses may includelung function, or cardiac rate, rhythm, andvariability. Techniques used to detect poten-tial biomarkers include bronchoscopy, nasallavage, or sputum induction. Techniques arealso available to measure airway size andmucociliary clearance rates using aerosolprobes and radiolabeled aerosol techniques.An additional approach is the use of nasalmasks or mouthpiece exposures of individual

subjects in place of an exposure chamber.These design features affect and often deter-mine the technologic and methodologicapproaches used for pollutant-generatedmonitoring and quantitative sampling (1).

There is a major difference with regard topollutant generation requirements betweenstudies utilizing facemask or mouthpieceexposures and those conducted in relativelylarge environmental chambers. With themouthpiece, a pollutant-air mixture must beproduced that only slightly exceeds the indi-vidual subject's respiratory intake require-ments, e.g., from 5 L min-1 to 50 L min-1with exercise. In contrast, for chambers oper-ating with a single pass (no recirculation), 5to 25 m3 min-1 is the likely flow rate require-ment, i.e., as much as 1,000 times greater.Usually the exposure duration required forthis greater generation capacity is also longerwith chamber studies than with mouthpieceor facemask studies.

Monitoring devices are used to determinewhether exposure levels in the breathing zoneare stable or changing. Ideally, monitors arereal-time devices calibrated to indicate con-tinuously the absolute pollutant concentra-tions. This level of performance can beachieved with certain pollutant analyzers suchas for ozone (03), but often the investigatoris provided only qualitative information bymonitors. In this situation, if quantitativemeasurements of pollutant levels are required,complementary analytical devices must beused that often are not real-time and onlyserially sample the exposure conditions.

The controlled clinical study provides theopportunity to examine both healthy volun-teers and individuals with underlying cardio-pulmonary diseases. Subjects typically areclassified by age, gender, race, lung function,and cardiovascular status. Normal volunteersare characterized by the absence of allergies,often documented by skin testing, the lack of

This article is part of the monograph on Environmentaland Occupational Lung Diseases.

Address correspondence to M.W. Frampton,University of Rochester School of Medicine, 601Elmwood Ave., Box 692, Rochester, NY 14642-8692USA. Telephone: (716) 275-4861. Fax: (716) 273-1114.E-mail: [email protected]

This work was supported by National Institutes ofHealth grants ES01247 and RR00044 and U.S.Environmental Protection Agency assistance agree-ments R826781-01 and R827354-01.

Received 29 February 2000; accepted 24 May 2000.

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hyperresponsive airways assessed by inhalationchallenge tests, and absence of hypertension orcardiac arrhythmias. Subgroups of healthyvolunteers may include adolescents, elderlysubjects, and smokers. Healthy volunteerstypically are able to perform vigorous exercisefor extended periods.

Exercise performed either on a treadmillor bicycle is an important component of theexposure study. Exercise enhances the pollu-tant dose both by increasing ventilation andby causing a switch from nasal to oralbreathing, effectively bypassing the nose.The nose may remove some particles andgases, variably reducing their delivery to thelower respiratory tract (2). In addition, theeffect of exercise on airway drying mayenhance the response to pollutants.

Finally, because dinical studies often usesmall numbers of volunteers, it is essentialthat sample size calculations be performed tomake certain the study has adequate power.This is often an important limitation in con-trolled human exposures. Statistical issues incontrolled clinical study designs have beenconsidered by Van Ryzin (3).

Assessing Responsesin Human StudiesThe choice of outcomes to be assessed inhuman clinical studies can be guided by bothscientific and practical considerations.Clearly, the outcomes of interest depend onwhat is known and expected about the effectsof the pollutant being studied. Results fromepidemiologic investigations, occupational oraccidental exposures, or animal exposurestudies may suggest the range of effects of agiven agent. The chemical behavior of theagent may predict its effects. For example,SO2 is a reactive and soluble gas and thereforeexerts its effects predominantly in the upperairways and major bronchi where it is com-pletely absorbed. 03, although very reactive,is relatively insoluble. Because it is notentirely dissolved in the epithelial lining fluidof the upper airway, it persists in the inhaledair, even to the alveolar space. Thus, effectsare expected and found throughout the respi-ratory tract. In contrast to these irritant gases,carbon monoxide passes into the blood withvirtually no pulmonary toxicity; its healtheffects result from binding to hemoglobinand resulting tissue hypoxia.

Practical considerations obviously guideand limit outcome measures in human dini-cal studies. For safety and comfort reasons,human studies utilize exposure protocols andpollutants designed to elicit transient, clini-cally insignificant effects in relatively healthysubjects, using minimally invasive measure-ment techniques. The usefulness of humanstudies has been extended with the develop-ment of techniques for safely sampling cells

of the lower airway and by the ongoingsearch for markers of pollutant effects. Thissection reviews traditional and more recentoutcome measures used in human clinicalstudies of air pollution.

Respiratory ResponsesVirtually every clinical test designed to assessthe presence or severity of respiratory diseasehas been used to measure the effects ofinhaled pollutants in either clinical or epi-demiologic studies. The use of traditionaltests of lung mechanics in human clinicalstudies has been reviewed (4), and is dis-cussed only briefly; emphasis instead is placedon more recently developed methods forassessing respiratory responses.

Pulmonary function tests. Despite thedevelopment of newer and more direct mea-sures of pollutant effects on the airways, sim-ple spirometry remains a mainstay in humanclinical studies. The measurement techniques,equipment, and interpretation have beenreviewed extensively (5). The most com-monly evaluated parameters obtained fromspirometry are the forced vital capacity(FVC), forced expiratory volume in 1 sec(FEV1), and the maximal flow-volume curve.During the past 10 years, the vast majority ofhuman pollutant exposure studies have evalu-ated responses with at least one of these para-meters. The reasons for selecting these testsare obvious. They are simple to perform, andthe results are reproducible within subjects.Standards for spirometry have been estab-lished by the American Thoracic Society (6),and ranges of normal values have been estab-lished (4). Results of testing, especially FEV1,have been found to correlate well with func-tional status. For example, in one study inhealthy subjects and in patients with chronicbronchitis, a reduction in FEV1 of more than5% in the healthy subjects and of more than15% in patients with airflow limitation wasrequired to be considered significant (8).

One potentially confounding problemwith spirometry is that the test itself may alterthe parameters being tested. For example, thedeep inspiration that precedes measurementsof forced expiratory flow causes transientbronchodilation and reduces inducedbronchoconstriction in the healthy individualbut may cause bronchoconstriction in theasthmatic. Performance of spirometry alsoappears to increase the airway production ofnitric oxide (9, a measurement used in somestudies to assess airway inflammation non-invasively. These changes should be consid-ered in designing and interpreting the resultsof clinical studies.

Other tests of pulmonary mechanicsindude the measurement of airway resistanceor its reciprocal, conductance, analysis ofpartial flow-volume loops and flow rates at

specific lung volumes, and closing volumes.Airway resistance testing is influenced bychanges in the major bronchi and upper air-way, including the larynx. Partial volumeflow rates and closing volumes were designedto provide more sensitive measures withgreater sensitivity for small changes, particu-larly in the more distal airways (10).Weinmann et al. (11) have used partial vol-ume flow rates to indicate possible delayedsmall airway effects following 03 exposure.Although these and other techniques mayprove more sensitive than FEV1 for detectingmild obstruction or peripheral airwaychanges, the methods often lack reproducibil-ity and there is uncertainty regarding theirinterpretation.

Airway responsiveness. The testing ofnonspecific airway hyperresponsiveness hasproven useful for assessing airway responses tolow concentrations of environmental airwaypollutants. These tests measure responses toinhalation of pharmacologic broncho-constricting agents such as methacholine, car-bachol, or histamine. Other stimuli ofbronchoconstriction used in clinical studiesinclude isocapnic cold air hyperventilationand inhalation of increasing concentrations ofSO2. Responses are measured with the usualpulmonary function measures, typically FEV1or airways resistance. Increasing concentra-tions of a bronchoconstricting agent areinhaled to construct a dose-response curve,and the results are expressed as the provocativeconcentration (PC) necessary to produce agiven change in function, such as a decrease inFEV, of 20% (PC20). The PC20 is obtainedfrom the log-dose-response curve by linearinterpolation of the last two points; the lowerthe PC20, the greater the responsiveness.

Asthma is characterized by a significantincrease in nonspecific airways hyper-responsiveness compared with that in healthysubjects. Studies of subjects with asthmatherefore need to use considerably lower ini-tial concentrations of the bronchoconstrictingagent. Furthermore, airway diameter influ-ences the results of challenge testing. Forexample, an airway already constricted to50% of its baseline diameter needs a muchsmaller concentration of methacholine toachieve a further 20% reduction. Thus, theresults of challenge testing are difficult tointerpret if bronchoconstriction is alreadypresent at the time of testing.

Airway challenge studies have been usedto assess the effects of pollutant exposure onairway responsiveness and to determinewhether baseline levels of responsiveness pre-dict lung function decrements in response topollutant exposure. For example, subjectswith mild asthma demonstrated increased air-way responsiveness following exposure toNO2 (12) or sulfuric acid aerosol (13).

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Healthy subjects experienced increasedresponsiveness to carbachol following 6-hrexposures to 2.0 ppm NO2, in the absence ofchanges in lung function (14). Utell et al.(15) found a relationship between baselineairways responsiveness assessed by carbacholin asthmatic subjects and responsiveness to aninhaled sulfuric acid aerosol. In contrast, air-ways responsiveness to methacholine was notpredictive of the FEV, decrement in responseto 03 exposure in either smokers ornonsmokers (16).

Fiberoptic bronchoscopy. Development offiberoptic bronchoscopy in the 1970s revolu-tionized the diagnostic evaluation of patientswith pulmonary disease, and this technique isnow widely used to sample the respiratorytract for research purposes. Its use in studiesof asthma has been reviewed recently (17).Bronchoscopy was first used by Seltzer andcolleagues (18) to detect airway inflammationin response to 03 exposure. Since then, bron-choscopy has allowed investigators to charac-terize the nature of the airway inflammatoryresponse to single and repeated exposures to03 in healthy, allergic, and asthmatic subjects(19-22). It has been used to study the effectsof exposure to many pollutants in addition to03, including NO2 (23-25), SO2, (26), acidaerosols (27-29), and diesel exhaust (30).A growing number of techniques have

been developed using the access provided bythe fiberoptic bronchoscope to sample fluidsand cells of the lower airway (Table 1).Bronchoalveolar lavage (BAL), the serialinstallation and removal of fluid through abronchoscope wedged in a distal airway, hasprovided an opportunity to sample the

epithelial lining fluid and cells of the distalairways. Evidence suggests that cells recov-ered by BAL reflect those present in the pul-monary parenchyma in disease states such assarcoidosis and idiopathic pulmonary fibrosis(31). However, cells from the pulmonaryinterstitium, some of which may have a keyrole in the pulmonary immune response(32), are not sampled using this technique. Amodification of the original technique of ser-ial instillation of aliquots of saline involvesseparate analysis of the first returned portionof fluid, which preferentially samples the dis-tal conducting airways in which the bron-choscope is wedged. Fluid obtained fromsubsequent aliquots more closely reflectsalveolar sampling (33). This concept ofregional lavage was extended with the use ofproximal airway lavage (34), in which acatheter with two inflatable balloons and aport between them is inserted through thebronchoscope into a mainstem bronchus.The balloons are inflated to isolate thebronchus, and repeated small-volume washesare performed using the port between theballoons. Care must be taken during the pro-cedure to avoid prolonged balloon inflation,as some subjects may experience significantlydecreased arterial oxygen tension duringocclusion of the airway. The technique hasbeen used successfully in both healthy volun-teers and subjects with mild asthma (35). Amodification of this technique has been usedto study the effects of 03 exposure in healthyand asthmatic subjects (36). However, thistechnique is not often used because of itsincreased complexity and variability as wellas difficulty in interpreting the findings.

The fiberoptic bronchoscope has beenused to instill particles into a single sub-segmental airway in human subjects to exam-ine localized epithelial responses in an isolatedlung region (36). The exposed segment isthen sampled subsequently using BAL and/orbiopsy. This approach is similar to segmentalallergen challenge in studies of the patho-physiology of asthma. It permits the deliveryof relatively high concentrations of particlesdirectly into the airway without the potentialrisks involved with inhalation exposures.

Epithelial cells and tissue can also beobtained through the bronchoscope usingbrushes or biopsy forceps, respectively.Endobronchial biopsies have been shown tobe safe in healthy volunteers and subjectswith mild or moderate asthma. Althoughbleeding can occur at the site of a biopsy, it isalmost never clinically significant. The tissuesamples are small, but sufficient material canbe obtained to examine cellular patterns ofgene expression using immunologic stainingand in situ hybridization. In studies utilizingpollutant and control exposures, with eachsubject serving as his/her own control, it isimportant to avoid carryover effects, in whichmucosal repair processes may influence theresults of the second study. The combinationof pollutant exposure and endobronchialbiopsies may induce subtle changes in airwaycytokine expression that persist as much asthree weeks after exposure (37).

Endobronchial biopsy should not beconfused with transbronchial biopsy, a proce-dure designed to obtain alveolar tissue fromthe human lung for clinical diagnostic pur-poses. The biopsy forceps are advanced into

Table 1. Bronchoscopic procedures used in clinical studies.

Procedure DescriptionBAL Bronchoscope wedged in a distal airway;

sequential instillation and recovery of sterile saline

Bronchial lavage Separate analysis of first recovered aliquot ofBAL (see above)

Proximal airway Double-balloon occlusion catheter placed in alavage main bronchus; lavage performed via port

between occluding balloonsBronchial brush Passage of small brush through suction port,biopsy brush airway surfaceEndobronchial Passage of tiny alligator forceps throughbiopsy suction port; obtain 1-2-mm tissue sample

from bronchial epithelium

Transbronchial Passage of tiny alligator forceps throughbiopsy the bronchial wall; obtain small samples of

alveolar tissue

Bronchial Agent ( allergen, particles) instilled via suctioninstillation channel of bronchoscope; sampling with BAL,

biopsy

Purpose and advantageSamples distal airway and alveolar space,with recovery of epithelial lining fluid,surfactant components, and cells;relatively reproducible findingsPreferentially samples distal conductingairways in comparison with subsequentlyrecovered aliquotsSamples large conducting airway separatefrom alveolar space and distal airways

Samples of airway epithelial cells; cellscan be cultured and passagedSamples epithelium in situ; providestissue for histology, immuno-cytochemistry, in situ hybridization

Samples alveolar tissue and distalbronchial epithelium

Most useful for studies of airway cellularresponses in vivo; allows safe study ofhigh tissue doses

DisadvantagesProtocols differ among labs; serialmeasurements not possible; findingsmay not reflect tissue effects

Sample is of mixed bronchial andalveolar origin; protocols differamong labsRelatively invasive; difficult toperform; may cause transienthypoxemiaLow viability and limited numberof recovered cellsLimited to major conducting airways;small tissue samples; relativelyinvasive; bleeding can occur(rarely significant)Small tissue samples; invasive;

Potential complications includepneumothorax, hemorrhageEffects of instillation may differfrom inhalation; difficult to controldose


UTELL AND FRAMPTON

the most distal airways and samples obtainedeither blindly or under fluoroscopic guidance.This procedure may be complicated by pneu-mothorax and rarely, lung hemorrhage, andhas not been used in clinical studies of airpollution. Recent studies have utilized trans-bronchial biopsy to demonstrate the presenceof inflammation at the alveolar level insubjects with nocturnal asthma (38).

Sputum induction. In recent years,sputum induction has emerged as a researchtool for sampling the cells and epithelial liningfluid of the lower respiratory tract in humans(39,40). Sputum can be reliably induced inasthmatic subjects as well as in most healthysubjects using nebulized 3-5% saline. Thecells recovered are representative of the lowerairways and provide a measure of airwayinflammation. Asthmatic subjects have moreeosinophils and polymorphonuclear leuko-cytes (PMNs) in sputum than healthy sub-jects. Sputum inflammatory cells increasedwith asthma exacerbations (41.42) and aller-gen challenge (43), and decreased with pred-nisone therapy (44). The results appear to bequalitatively similar to findings in the first(bronchial) aliquot ofBAL fluid (45).

The process of sputum induction itselfappears to induce a mild airway inflammatoryresponse. For example, when two inductionsare performed 24 hr apart, the second induc-tion has a higher recovery of PMNs (46,47).Thus, caution must be used in designingstudies with serial measurements. Sputuminduction is well tolerated, even in those withsevere asthma, but some asthmatic subjectsexperience bronchoconstriction in response tothe nebulized saline. Pretreatment with bron-chodilators is effective in preventing theaerosol-induced bronchoconstriction.

Some subjects are unable to producesputum following induction. Approximatelytwo-thirds of healthy subjects, and more than90% of asthmatic subjects, are able to beinduced. Individuals able to produce sputumdo so on subsequent challenges and the find-ings on repeat sputum inductions are quiteconsistent (r= 0.80) among individuals (48).Fahy et al. (49) demonstrated that sputuminduction detects airway inflammation fol-lowing exposure to 0.4 ppm 03 for 2 hr inhealthy subjects. In contrast, exposure to 0.3ppm NO2 for 1 hr, with moderate intermit-tent exercise, did not alter cell recovery ininduced sputum in subjects with mild asthmaor chronic obstructive pulmonary disease(COPD) (50). These findings are consistentwith findings in BAL fluid following expo-sures to 03 and to NO2. Sputum inductiontherefore holds promise as a noninvasivemethod for assessing airway inflammation indinical and field studies.

Markers in exhaled air. Biochemicalprocesses at the epithelial level may release

gaseous products, some of which can bedetected in the exhaled breath. Investigatorshave sampled exhaled air for a variety of sub-stances as markers of either airway inflamma-tion or injury, including hydrogen peroxide(51), CO (52, isoprene (53), ethane (54), andpentane (55). The technology has been devel-oped to measure concentrations of over 100volatile chemicals in the exhaled breath, withpotential applications in field studies of envi-ronmental exposures (56). Foster et al. (53)found small but significant increases in exhaledisoprene levels in healthy subjects followingexposure to 03 at 0.15-0.35 ppm for 130 min.

Measurements of exhaled nitric oxidehave attracted wide interest as a means ofdetecting lung inflammation (57). NO is pro-duced from the action of nitric oxide synthase(NOS) on L-arginine. A variety of cellsexpress one or more of the three isoforms ofNOS, including airway epithelial cells andendothelial cells. Concentrations ofNO areincreased in the exhaled air of people withasthma compared with healthy subjects (58).Mild asthmatics not requiring inhaled or oralcorticosteroids have exhaled NO levels 7-foldgreater than normal subjects (59). NO levelsincrease further with clinical exacerbations,correlate with the degree of airway hyperre-sponsiveness in steroid-naive asthmatics (60),and decrease following therapy with corticos-teroids (61,62). A preliminary report (63)indicates that exhaled NO is increased inhealthy subjects following exposure to 0.25ppm 03 2 hr daily for 3 days, suggesting NOmay prove to be a useful marker of pollutant-induced inflammation. However, the highconcentrations ofNO found in the pharynxand nasal passages can confound the measure-ment of NO production by the lower air-ways. Furthermore, the lungs have a very highdiffusing capacity for NO, and most studieshave not taken into account the removal ofNO from the airways by capillary blood (64).

The measurements of gases and volatileorganic molecules in exhaled air show promiseas noninvasive markers of pollutant effects,but considerable work remains to understandthe significance of the observed changes.

Cardovasculr Re"ponsesUntil recently, concern about possible cardio-vascular effects of exposure to air pollutionwas limited almost exclusively to CO becauseof the known detrimental effects of CO onoxygen delivery. Few studies have assessedcardiovascular effects of other pollutants. In1985 Linn and co-workers (65) found smallreductions in systemic blood pressure inhealthy subjects exposed to NO2, but thefinding has not been confirmed in subsequentstudies. Drechlser-Parks (66) used a non-invasive impedance cardiographic method toestimate changes in cardiac output following

2-hr exposures of healthy subjects to 0.60ppm NO2, 0.4 ppm 03, and a combinationof NO2 and 03. Compared with air expo-sure, cardiac output was reduced followingexposure to the combination ofNO2 and 03,but not for the individual pollutants.Changes in blood pressure were not reported.Gong and co-workers (67) performed per-haps the most definitive study of cardiaceffects of 03 exposure. Men with and with-out stable hypertension underwent arterialand right heart catheterization, and then per-formed intermittent exercise in 0.3 ppm 03for 3 hr. No differences from air exposure wereseen for a variety of indices of cardiac function.However, heart rate, the rate-pressure product(an index of left-ventricle work), and thealveolar-arterial gradient in oxygen tension allshowed greater increases following 03 butnot air exposure. This combination of effectscould be clinically important in individualswith critical coronary lesions.

Epidemiologic studies of ambient partideshave shown a surprisingly robust associationwith cardiovascular events, but no mecha-nism has been found to explain the relation-ship. Proposed hypotheses explaining cardiaceffects of inhaled particles have includedhypoxemia (68), direct effects of particles ortheir reaction products on myocardial cells(69), and cardiac consequences of airwayinflammation or injury (70,71). Recentstudies in healthy and compromised animalsfrom the laboratories of the U.S.Environmental Protection Agency (69) andfrom Harvard University (Boston, MA) (72)have suggested that inhalation of particulatepollutants may induce changes in cardiacrhythm or repolarization. Pope and co-work-ers (68) used pulse oximetry to study panelsof elderly subjects residing at elevation. Theyfound no changes in oxygen saturation, butthere were small but significant increases inheart rate associated with outdoor particleconcentrations. A 100-pg/m3 increase in theprevious-day level of particulate matter < 10pm in diameter (PM1O) was associated with a

95% increase in the odds of a 10-beatincrease in the heart rate. These observationshave intensified efforts to understand the rela-tionship between pollutant exposure andchanges in cardiac function.

One tool being used in this effort iscontinuous cardiac monitoring, or Holterrecording. Originally used to identify silentarrhythmias in cardiac patients, cardiac moni-toring has revealed that both the pattern ofvariation in heart rate, or heart rate variability,and the pattern of electrical repolarization ofthe heart are markers of cardiac health or dis-ease. Decreased heart rate variability as well asabnormalities in the duration, dynamics, andheterogeneity of repolarization are establishednoninvasive predictors of arrhythmic events in


HUMAN EXPOSURES

patients with cardiovascular diseases (73-77)and in healthy subjects (78). Dispersion ofrepolarization, a noninvasive electrocardio-graphic (EKG) measure of spatial heterogene-ity of repolarization, was increased in patientswith COPD (79), but also in healthy subjectsin response to hypoxemia (80).

These observations suggest that detailedanalyses of the continuous ambulatory EKGfor specific parameters, including heart ratevariability and repolarization, might provideinsights into the nature of the cardiac effectsof pollutant exposure.

Pharmacokinetics ofInhaled VaporsAlthough inhaled chemicals such as NO2, 03,and SO2 may cause direct damage to airwayand lung tissue, other vapors diffuse veryrapidly from the alveolar spaces, through thelung tissue, and into blood, causing no directlung injury. CO diffuses into the blood andbinds avidly to hemoglobin. Other volatilechemicals are absorbed by inhalation and sub-sequently cause toxicity at sites distal to the res-piratory tract. In addition, with some of thesechemicals, it is not the parent compound that ismost toxic but the metabolites, which may bemore reactive and more damaging to the tissue.

The ultimate objective of many toxicologicstudies is to take results from animal studiesand attempt to predict or describe expectedhuman responses. Physiologically based phar-macokinetic (PBPK) models are used in anattempt to describe the overall disposition ofan inhaled chemical by simulating the uptake,distribution, metabolism, and elimination ofthe inhaled material (81). The PBPK model isthen used in extrapolation between speciesbased on animal scaling techniques. The ade-quacy of interspecies models should be testedby extrapolating the results of experimentswith animal models to humans and comparingthem with the relatively limited data availablefrom controlled human exposures.

Although the strengths and limitations ofthe PBPK approach are beyond the scope ofthis article, Gargas and Andersen (81) previ-ously reviewed the necessary parametersrequired to develop such models as well as theapplication to several chemicals. Such modelshave been developed for styrene (82) compar-ing data for rats and humans. The extrapo-lated model performed very well, predictingthe concentration profile in humans exposedto 80 ppm styrene. Utell and co-workers (83)examined the pharmacokinetics of cyclicsiloxanes in quantitative controlled exposurestudies. Silicones are commonly found in per-sonal care products including antiperspirantsand hair sprays, with potential for exposureby the respiratory route. Octamethlycyclo-tetrasiloxane (D4), a volatile siloxane, is amajor silicone component of these consumerproducts and as many as 200 million

Americans are exposed to D4 in personal careproducts on a daily basis.

In these studies, 12 healthy subjectsinhaled 10 ppm D4 (122 pg/L) or air (con-trol) during a 1-hr exposure via a mouthpiece.Inspiratory and expiratory D4 levels were con-tinuously measured as well as exhaled air andplasma levels before, during, and after expo-sure. Mean D4 intake was 137 mg, with amean uptake of 13 mg (intake x depositionfraction). The mean deposition fraction at restwas 13% and with exercise it fell to 7%.Plasma measurements of D4 gave a meanpeak value of 79 ng/g and indicated a rapidnonlinear blood dearance. These types of dataregarding the mean D4 deposition fractionand uptake are applicable to estimates of expo-sure from both consumer product use andoccupational exposure to D4 vapor.Subsequent studies using 14C-labeled D4should allow even better quantitative assess-ments and detection of metabolites in bloodand urine. Comparison with data from ongo-ing animal studies (84,85) will allow develop-ment of PBPK models for extrapolation tohumans. The data from controlled humanstudies should contribute in a meaningful wayto the risk assessment process.

Studying Susceptible SubjectsThe concept of susceptibility is highly relevantto public health protection and to the deliv-ery of health care. The United States CleanAir Act (86) mandated that national ambientair quality standards be established to protectthe health of all susceptible groups within thepopulation.

There is a high degree of variability inhuman responses to environmental agents. Forexample, decrements in FEV1 following 4-hrexposures to 0.22 ppm 03 varied from 0 tomore than 50% (Figure 1) (16), and yet thedeterminants of this variability are largelyunknown. Some of the known and postulatedfactors influencing variability in humanresponses to pollutants are as follows: age, gen-der, body size, exercise, pre-existing disease,atopy, infection, airway geometry, smoking,pregnancy, exposure history, airways respon-siveness, nutritional status, antioxidant vitaminintake, and antioxidant enzyme expression.Variability is generally increased in populationswith respiratory disease, especially asthma.Some of the observed variability may be relatedto the actual dose delivered to the lung epithe-lium or to the distribution of the inhaled pol-lutant. For example, retention of insoluble2 pm particles was increased 50% in subjectswith COPD compared with healthy subjects,and the increased deposition correlated withthe severity of obstruction (87). The diversityof human responses and responsiveness mustbe considered in designing and conductinghuman clinical studies of pollutant effects, and

careful attention must be given to subjectselection and characterization.

The concept of susceptibility is often over-simplified by both researchers and regulators.Rather than a single characteristic of a givenpopulation, susceptibility varies by pollutantand by the health effect being considered.There may be multiple health effects of pollu-tant exposure (i.e., symptoms, lung functiondecrements, airway inflammation, infection,cardiac effects, etc.), with a number of suscep-tible populations for which mechanisms andsusceptibility factors differ. Susceptibility for agiven health effect may not confer susceptibil-ity to a different health effect. A strikingexample is exposure to O3: individuals whoexperience the greatest reductions in lungfunction are not necessarily more likely toexperience airway inflammatory effects(19,35). Furthermore, the mechanisms bywhich pollutant exposure contributes to respi-ratory mortality may differ from those respon-sible for excess cardiovascular mortality.

An understanding of the mechanisms bywhich ambient concentrations of pollutantsincrease mortality will require human clinicalstudies of susceptible subjects. However, suchstudies are limited by the special needs andchanging health status of the subjects.Increased variability in measured parametersoften requires that studies of susceptible sub-jects have larger numbers of subjects perstudy group compared with studies of healthysubjects. Medication use may alter responsesin some subjects. Measures must be taken toprotect the safety of susceptible subjects, whomay be more vulnerable to adverse effects orcomplications from testing procedures orpollutant exposures.

In this section we focus on the increasedsusceptibility conferred by three chronicconditions: asthma, COPD, and cardio-vascular disease.

10-

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-30-

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-50-

-60 IBaseline 2 hr 4 hr

Figure 1. Percent change in FEV, during and afterexposure to 0.22 ppm ozone, with intermittent exer-cise, in healthy nonsmoking subjects who were eithernonresponsive (change in FEV, < 5%; *) or responsive(change in FEV, > 15%; a) to ozone. Data from Torresetal. (19).


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AsthmaIn clinical studies, asthmatics exhibit exagger-ated lung function responses to SO2, acidicaerosols, and in some studies, NO2 and 03.Asthmatics may also experience an enhancedairway inflammatory response to 03.

In the laboratory, the most striking effectof acute exposure to SO2 at concentrations of1.0 ppm or below is the induction ofbronchoconstriction in asthmatics after expo-sures lasting only 5 min (88). In contrast,inhalation of concentrations in excess of 5.0ppm causes only small decrements in airwayfunction in normal subjects. Similarly, clini-cal studies have identified exercising adoles-cent asthmatics (89) and adult asthmatics(90) as susceptible to sulfuric acid aerosols athigh ambient concentrations, levels that donot affect healthy volunteers. Although sev-eral controlled human studies have foundasthmatics responsive to low levels of NO2,the findings have not been consistent (91).The conflicting results among these studiesare probably related to differences in subjectselection and exposure protocols.A number of epidemiologic studies have

suggested that emergency room and hospitalvisits for asthma are increased on high-03days. It is therefore surprising that most con-trolled clinical studies generally have notfound striking differences in lung functionresponses to 03 in asthmatic compared withhealthy subjects. Several possible explanationsexist. In contrast to studies with healthy vol-unteers, few studies of asthmatic subjects havebeen performed using prolonged exposures orrepeated daily exposures. Furthermore, fewstudies with asthmatic subjects have incorpo-rated multiple periods of moderate-to-intenseexercise, a factor that contributes to changesin airway function with low-level 03exposure in healthy volunteers.

One study has addressed some of theseissues. Horstman and colleagues (92) exposed17 subjects with clinically active asthma and13 healthy subjects to 0.16 ppm 03 for7.6 hr, with multiple prolonged periods ofmild exercise. As shown in Table 2, asthmaticsubjects had significantly larger decrements inFEV1 and in FEV1/FVC, despite occasionaluse of bronchodilators before and during theexposure. This study suggests that peoplewith clinically active asthma may be atincreased risk for bronchoconstriction follow-ing prolonged exposures to environmentallyrelevant concentrations of 03.

Two recent studies suggested that mildasthmatics may experience a neutrophilic air-way inflammatory response to 03 exposurethat is more intense than in healthy subjects(22,93). Recent studies have also shown that,in asthmatics, 03 exposures at concentra-tions sufficient to induce an airway inflam-matory response increase responsiveness to a

subsequent allergen challenge (94). Clinicalinvestigations therefore suggest that themechanisms for exacerbation of asthma fol-lowing 03 exposure indude bronchoconstric-tion, worsening of airway inflammation, andincreased responsiveness to allergen challenge.

Asthmatics may also be more sensitive tocombinations of pollutants. Frampton et al.(95) examined the effects of prior exposure tolow-level sulfuric acid aerosol on the airwayresponse to 03 in healthy and asthmatic sub-jects. Exposure-response relationships wereexamined using three levels of 03, 0.08, 0.12,and 0.18 ppm. The exposures were preceded24 hr earlier by exposure to 100 pg/m3H2S04 or NaCl aerosol. The acidic aerosoland oxidant exposures were 3 hr in duration.Thirty healthy and 30 allergic asthmaticswere studied. The findings revealed an inter-active relationship between the 03 exposureconcentration and H2S04 or NaCI aerosolpreexposure in asthmatics but not in healthysubjects. For the asthmatic subjects, 03concentration-related differences in lungfunction were observed with H2S04 preexpo-sure but not with NaCl preexposure. Theseeffects were observed for both FVC and FEV,immediately and 4 hr after 03 exposure.These data suggest that preexposure toH2S04 aerosols may alter responses to 03 inexercising asthmatics.

Chronic Obstructiv Pulmonary DiseaseEffects of inhaled pollutants in COPD havenot been extensively examined. Patients withCOPD demonstrated similar or decreased lungfunction responsiveness to 03 at levels up to0.30 ppm compared with healthy nonsmokersof comparable ages (96). To determine if low-level NO2 induces changes in pulmonary func-tion, Morrow et al. (97) investigated responsesto inhalation of 0.3 ppm NO2 for 4 hr in 20COPD subjects with a mean age of 60 years,and all with a history of cigarette smoking.These subjects were compared with 20 elderlyhealthy subjects. Criteria for indusion includeddyspnea on exertion, airways obstruction[FEV1/FVC = 0.58 ± 0.09 (SD)] and a lack ofresponse to inhaled bronchodilators. Duringintermittent light exercise breathing NO2,COPD subjects demonstrated progressivedecrements from baseline in FVC and FEV,and no change with air. Subgroup analyses sug-gested that responsiveness to NO2 decreasedwith increased severity of the COPD.

Cardiovascular DiseaseStudies of low-level exposure to CO havefocused on subpopulations with ischemicheart disease and peripheral vascular disease.In patients with exertional angina, early onsetof angina pectoris and depression of theST-segment on the electrocardiogram (amarker of myocardial ischemia) have beenconsistently observed at carboxyhemoglobin(COHb) levels of 2-4% by several investiga-tive teams. In the largest of these studies, theHealth Effects Institute Multicenter COStudy (98), 5 and 12% decreases in the timeto onset of ST-segment depression wereobserved at COHb levels of 2 and 4%,respectively. Significant decreases in time toonset of angina were also demonstrated atthese COHb levels. These end points areremarkably consistent and are compatiblewith the hypothesis that an elevated COHblevel impairs the response of the myocardiumto increased metabolic demands.

New Experimental ApproachesConcentated Ambient Air PartidesAmbient air contains a complex mixture ofparticles and gases containing a variety ofchemicals at trace levels that may interact incausing health effects. A technology recentlydeveloped allows fine particles in the air to beconcentrated in real time. Air is drawnthrough a series of virtual impactors designedso that particles less than 2.5 ,um in aerody-namic diameter are progressively concen-trated up to 25- to 30-fold (99). Ambientgases, and particles in the ultrafine size range(< 100 nm diameter) are not concentratedusing this methodology.

This instrument has been used to demon-strate effects of ambient particulate matter inanimals. For example, Gordon and col-leagues (100) exposed rats to concentratedambient air particles (CAPs) or filtered airfor 3 hr, and found a significant but transientincrease in circulating PMNs along withsmall alterations in heart rate associated withCAPs exposure. Godleski and colleagues (72)exposed 12 dogs, 6 of which had surgicallyplaced devices to induce transient coronaryocclusions, to CAPs or filtered air on multi-ple occasions. In the 6 surgically treateddogs, exposure to CAPs was associated with ashortened time to ST elevation and anincreased magnitude of ST elevation during

Table 2. Lung function changes following exposure to 0.16 ppm ozone for 7.6 hr.aAsthmatic Nonasthmatic

Air Ozone Air Ozone pbFVC M% change) -0.3 ± 1.5 -12.2 ± 3.1 0.5 ± 0.9 -8.3 ± 1.8 0.33FEV1 (% change) 2.7 ± 3.0 -16.7 ± 4.2 1.2 ± 0.8 -8.6 ± 1.9 0.04FEV1/FVC(%) 2 ± 1 -4±2 0±1 -1 ±1 0.02"All values represent postexposure minus preexposure, means ± SE. hp-value: asthmatic vs nonasthmatic. Data from Horstman et al. (92).


HUMAN EXPOSURES

coronary occlusion. The implications ofthese studies for human health remainunclear, and human studies using CAPs arenow in progress (101,102).

The key strength of this technology is theability to separate particle effects from gaseouspollutant effects in the atmospheric mix.However, there are several potential problems.The actual exposure concentration and parti-de composition may vary day to day and evenhour to hour, depending on changes in out-door particle levels and sources during theexperiment. The limited ability to define thechemistry of the CAPs, and the day-to-daychanges in particle composition, introduces anadditional element of variability into theexperimental design. In addition, the failure toconcentrate ambient ultrafine particles mayhave significance. Animal exposure studiessuggest that ultrafine particles have increasedpotential to induce an airway inflammatoryresponse when compared on an equal massbasis with larger partides, perhaps because oftheir greater number and surface area (103).The relative absence of ultrafine particles andgaseous pollutants must be considered wheninterpreting the results ofCAPs studies.

Diesel Engine E ustAnother approach to the problem of mixturesis illustrated by recent studies of exposure todiesel exhaust. Diesel engines have gainedfavor in may parts of the world because ofreduced operating costs and reduced emis-sions of CO, CO2, and hydrocarbons, com-pared with gasoline engines. However, theyappear to release greater quantities of fine andultrafine partides and nitrogen oxides (104).

Salvi et al. (30) exposed healthy subjects for1 hr to dilute exhaust from an operating Volvodiesel engine in a specially designed exposurefacility. Bronchoscopy with bronchoalveolarlavage and bronchial biopsy were performed 6hr after exposure. In comparison with controlair exposures, a significant increase wasobserved in epithelial leukocytes and mastcells, with increased expression of the adhesionmolecules ICAM-1 and VCAM-1 in submu-cosal endothelium. Thus, diesel exhaustinduces airway inflammation in healthy sub-jects in the absence of effects on lung function.

Nasal challenge with diesel exhaust particleshas been shown to enhance the local ragweed-specific IgE response in subjects with allergicrhinitis and to drive the nasal cytokine responsetoward the Th2, or allergic phenotype (105).These findings raise concerns about dieselexhaust as a contributor to the increased preva-lence of allergic rhinitis and asthma.

"Smokinge' MachinesUsing smoking machines, controlled expo-sure studies have examined the role of envi-ronmental tobacco smoke (ETS) in the

exacerbation of asthma (106). Of course, bydesign, participants cannot be blinded to theETS exposure. Typically, brief exposure toETS produces symptoms such as eye andnasopharyngeal irritation. Although there area few positive studies with ETS, the majoritydocument no significant effect on FEV1 ormeasures of airway responsiveness. However,study design has not been optimal; limitedsample size, brief duration of exposure, andunrealistic exposure designs all emphasize theneed for careful protocol development if thisimportant clinical and social issue is to bebetter understood.

Model Partide EiposuresStudies of CAPs, diesel exhaust, and otherreal-world particles are unable to determinewhich particle characteristics, or combina-tions of characteristics, are responsible for theobserved effects. One approach to this prob-lem is to use particles with specific size char-acteristics or chemical composition to testspecific hypotheses. This approach permitscareful control of the particle to be tested,and permits precise exposure-response assess-ments. However, the particle design must beguided by detailed chemical and size charac-terization of ambient particles and by prelimi-nary animal exposure studies to assure safety.

Our laboratory is using this approach totest the hypothesis that particles in the ultra-fine size range have greater potential thanlarger particles to induce airway inflamma-tion, systemic acute phase responses, andchanges in cardiac repolarization. We havedeveloped and tested a facility that permitsexposures of humans to ultrafine particles ofvarying composition and that also permits thequantitative determination of exposure levels,respiratory intakes, and depositions of theaerosol. Using this facility, we have initiatedclinical studies of exposure to ultrafine parti-cles in healthy human subjects at rest andexercising (107).

Conclusions and FutureDirections for Human StudiesControlled dinical studies provide a means forexamining responses to air pollutants andchemical vapors, especially those identifiedfrom epidemiologic studies. Well-characterizedexposures have been performed either inenvironmental chambers or by mouthpieceand the responses assessed primarily by respira-tory mechanics or from direct sampling of res-piratory tract fluids, cells, and tissues. BALstudies have demonstrated pollutant-inducedinflammation, lung injury, and decreased hostdefense capacity. Less invasive techniques suchas nasal lavage and induced sputum also havebeen used to show pollutant-induced inflam-mation. Clinical studies have used a widerange of potentially sensitive subpopulations

induding asthmatics, children, the elderly, andpeople with COPD and coronary artery dis-ease. To date, clinical studies with air pollu-tants have identified susceptible populations,characterized exposure-response relationships,and examined lowest-effect levels. With recentepidemiologic observations linking elevatedpartide concentrations with adverse cardiovas-cular events, clinical studies will focus onmarkers of systemic effects and changes in car-diac function linked with acute cardiac events.The introduction of the particle concentratorshould strengthen the ability of clinical studiesto study real ambient mixtures.A variety of opportunities exist for exten-

sion of clinical studies into new arenas.Zelikoff et al. (29) examined effects of H2S04aerosol exposures, comparing responses in alaboratory animal model commonly used fortoxicologic assessment with those observed inhuman volunteers. The purpose of this collab-orative study was to provide a basis for extrap-olation of findings in animals to humans.Substantial concordance between humans andrabbits was found with regard to the cellsrecovered in BAL fluid, and alveolarmacrophage immuno-responsiveness, in rela-tion to H2S04 exposure. For example, in bothspecies, a single 2-hr inhalation exposure toH2S04 aerosol failed to evoke an inflamma-tory response, alter lavageable protein levels, orproduce changes in cell viability. The results ofdirectly comparative studies can provide datanecessary for risk assessment and for predictingthe accuracy of extrapolation modeling.

Finally, there are unique opportunities tocombine the strengths of clinical and epi-demiologic studies in the effort to understandmechanisms of susceptibility to pollutanteffects. Hackney et al. (108) recognized theusefulness of this approach, and in a very lim-ited study, found that four healthy subjectsresiding in Canada showed greater decre-ments in FEV1 following laboratory expo-sures to 03 than did four residents ofsouthern California. This was considered tobe evidence that chronic exposure to 03 inCalifornia blunted responsiveness in thosesubjects, although the number of subjects wastoo small to derive any definite conclusions.A parallel approach may prove useful in

determining the mechanisms involved inresponses to particle exposure. For example,groups of subjects identified from an epidemi-ology or panel study as susceptible (symp-toms, lung function, medication use, heartrate variability, etc.) or nonsusceptible to par-ticle effects could be studied in the laboratoryunder controlled conditions, either to con-centrated ambient particles or to a model par-ticle. Conversely, groups of individualsdetermined to be responders in a clinicalstudy could be followed in a panel study todetermine whether laboratory responses were


UTELL AND FRAMPTON

reproduced with actual ambient exposures.These approaches will require a new level ofcollaboration between investigators withexpertise in epidemiology and human clini-cal studies but may provide key clues in ourunderstanding of the mechanisms forsusceptibility to pollutant effects.

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