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Enhanced Response to Ozone Exposure duringthe Follicular Phase of the Menstrual CycleSusan D. Fox,' William C. Adams,1 Katherine A. Brookes,1 and Bill L.Lasley2IHuman Performance Laboratory, Department of Physical Education and 2Department ofReproduction, School of Veterinary Medicine, University of California, Davis, CA 95616USA

Acute ozone (03) exposure has been shownto result in short-term airway inflammationin both animals and humans (1,2). Thisinflammatory response involves both theinfiltration of neutrophils (1-3) and therelease of cyclooxygenase products fromarachidonic acid in the lung and airways(3). Seltzer et al. (3) observed significantincreases in the concentrations of pro-staglandins E2, F2a, and thromboxane B2(cyclooxygenase products) in bronchoalveo-lar lavage fluid after exposure to 03. Inaddition, prostaglandins E2 and F2a appearto stimulate pulmonary neural afferents ini-tiating tachypnea and cough, which areresponses characteristic of acute 03 expo-sure (4,5). Further, Schelegle et al. (6)found that pretreatment with indometh-acin, a nonsteroidal anti-inflammatorydrug, significantly attenuated 03-induceddecrements in forced vital capacity (FVC)and forced expiratory volume in 1 sec(FEV1). These findings imply that therelease of prostaglandins and subsequentinflammation in the lung and airways con-sequent to acute 03 exposure are involvedin routinely observed pulmonary functiondecrements.

Progesterone inhibits prostaglandinproduction in the uterine endometrium,though this inhibition fluctuates with thevariant changes in progesterone concentra-tion observed throughout the female's men-strual cycle (7,8). If cyclical changes insteroidal hormones alter a woman's re-sponse to uterine inflammation, could fluc-tuations in these hormone levels alter indi-vidual responses to a known respiratoryinflammatory inducer (viz, 03)? A hy-pothesis not yet investigated is that expo-sure to 03 during the follicular (F) men-strual cyde phase, when progesterone levelsare at their lowest, might result in enhancedresponses due to the absence of anti-inflam-matory influences of this steroid. The pur-pose of this study was to determine ifyoung, adult females respond differentially

to 03 inhalation with respect to menstrualcycle phase and progesterone concentration.To investigate this hypothesis, healthy,young, adult females were exposed to 0 inthe F and mid-luteal (ML) phases of tLeirmenstrual cycles.

Nine healthy, young, adult females,who participated in recreational aerobicexercise on a regular basis, served as sub-jects. We screened each woman for dini-cally normal pulmonary function, absenceof significant allergies, abstention from tak-ing birth control pills, and normal menstru-al function [intermenstrual interval ofgreater than 26 days, with sustained rise ofpregnanediol-3-glucuronide (PdG) greaterthan 3 pg/mg creatinine for more than 4days during the luteal phase]. All subjectswere nonsmokers and had not resided in anarea of high air pollution within the previ-ous 6 months.

Before initiating experimental proto-cols, each subject completed an orientationsession in which baseline pulmonary func-tion was obtained and specific equipmentand requirements of the study were re-viewed. We informed subjects of the pur-pose, procedures, and potential risks ofparticipation in the study before theysigned an informed consent form approvedby the University of California's HumanSubjects Review Committee. After com-pleting the experimental protocols, weassessed basic anthropometry, includingbody composition determination via hy-drostatic weighing, and maximum oxygenuptake (VO max). These results are pre-sented in Table 1.

We studied subjects throughout two tofour complete (though not necessarily con-secutive) menstrual cycles. Early morningurine samples (3 ml) were collectedthroughout the study period and analyzedfor estrone conjugates (E1C) and PdG(which were both indexed by creatinineconcentrations of the same sample to adjustfor variations in urine volume) to document

Table 1. Group anthropometric, V02max, and pulmonary function data

Height(cm)

165.4±4.9160.0175.3

Weight Fat V02max V max(kg) (% body wt) (mVkg/min) (I/min)

64.5±8.3 26.2±4.7 45.3±4.2 111.4±17.952.1 19.2 40.9 84.778.8 34.1 54.7 140.6

FVC(I)

4.0±0.443.24.7

FEV1(I)

3.4±0.462.94.4

Exosr to. oon.e (0:),3)a itoic comonetofphotocemical'sm g,resulsinsignfiat

airwayl nlamm6tion, re'p'tr dfort, ..and- pumonary n nm..unction m ment.

..~ ~~~..These: eflWcca be redce via pretreat:ment.with anti4n sndo- agents ..Pro-.gesterone, a gona.d.al stero"id, is known torduce geineral ~infamto nthe'.uterine:..W.d.W........nen:|ni.t e.nenoetim. Hoevr,iti not :.kn-ownm*-.- .......

wher ft ian blood levels of prog-esterone.. which ae Iexpeinced durin`hg thenormal femalemenstrual cyle could.alter03 ia:...:::....:mmator-induced pulmona re-sponses. In this:study, we tested he hy-pothesis that young, adult females are. .moreu . ...... ..o ii nresonsveo 0in alainwithrspc to

:pulmona:ry u-nction, impairment duringtheir follicular (F) menstrual phase when*pprogstrne.levels are lowet.than durig.P

., F;E 9 ..... .. .. i..their md-luteal (ML) phase when proges-terone leves are highes NNine subject withnormd. ovarian fu.niction were exposed inrandom order for1& hr eacto filtered airand t0....3. ppm. 0 in their F.d MLmenstual pha'ses (zone responsivenesswas measured by percent change in pul-monary fon om - o postexposure.Significat gs cnc i ffects (filteredair vensus 03) were observed for forced vitalcapacity (FVC), forced expiraory volume inI sec. .EY1), and frcd pirator flowbetween.25 and 75% of FVC (FEF2 75;p. .05). . More importandy,. the pulm yfunction.'Rflow .rates, FEY a.ndFEF1275,showed a:: significt mentrual phae -andgas concentration interaction effect, withlarger decrements..observed...n the F men-strua phae whn tne concentm-tions were s t lowert. We concudethat young, adult -females appear to be morrespnv to ac.u..e..0 during. the

F phase dtha durng se ML phas of theirmenstral cycles. This difference 'in pul-monary. function response could be relatedto the'an-inQaflammatory effec. of in dprogeste.irone conicentratio"ns during thelusal phas. Key uwrds: air pollution; forcedepirato. y flow rates; pulm uoarynctionimpairment, stetoid hormoones. EnvironHcaoh Perspect i01:242-244(1993)

a normal hormonal profile. We random-ized subjects and exposed them to either fil-tered air or 0.30 ppm 03 in the F and MLphases of ovulatory menstrual cydes. Withday 0 representing the day of the periovula-tory estrone conjugate peak, the F phase wasdetermined to be days -6 through the first

Address correspondence to W. C. Adams, Depart-ment of Physical Education, University of Califor-nia, Davis, CA 95616 USA. This study was sup-ported by the State of California Air ResourcesBoard (Interagency Agreement A933-096) and bythe Superfund Basic Research Program(P42ES04699). The authors gratefully acknowl-edge the technical assistance of Richard Fadling,the laboratory assistance of Robin Looft, TaraMcKittrick, and Kerri Winters, and SusanShideler, who contributed help with ovarian hor-mone analysis and data interpretation. Sincereappreciation is extended to the subjects for theircontribution of considerable time and effort.

Environmental Health Perspectives

Means ± SDMinimumMaximum

Age(years)

26.9±5.32034

Abbreviations: V02max, maximum 0 uptake; VEmax, maximum minute ventilation; BTPS, body tempera-ture, pressure, saturated; FVC, forceWvital capacity; FEV1, forced expiratory volume in 1 sec.

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day of menses. The ML phase was similarlycalculated as day +5 to +10. The exposureprotocol consisted of exercise work ratessuch that ventilation minute volume (VE)was about 50 1/min BTPS (body tempera-ture, pressure, saturated). The group meantotal inhaled dose of 03 during the 0.30ppm 03 exposures did not differ significant-ly between the F (859.9 ppm-1) and ML(855.6 ppm-1) phases. We conducted allexposures in a moderate ambient environ-ment with constant airflow provided from afloor fan. Subjects were not told whetherthey received 03.

We obtained pulmonary function mea-surements immediately before and aftereach experimental protocol. At least tworepeated maneuvers of forced expirationafter maximal inspiration were obtainedusing a Collins Modular office spirometer(model 3000). An on-line data acquisitionsystem, which interfaced the spirometermodule linear potentiometer output volt-age (associated with lung volume changes)and an analog-to-digital converter for read-ing into a Digital Equipment Corp. LSI11/2 microcomputer, was also used. Pul-monary function on-line computer deter-minations included measurements of FVC,FEV1, and forced expiratory flow between25 and 75% of FVC (FEF2,-75). In addi-tion, we monitored VE and heart rate dur-ing each protocol.

Subjects inhaled air mixtures duringexperimental protocols via a blow-by oblig-atory mouthpiece system, described indetail previously (9). Inspiratory 03 con-centrations in the mixing chamber weremonitored continuously by samples drawnthrough 0.64-cm inner diameter Teflontubing connected to a Dasibi 03 meter.The digital reading of 03 concentration inppm was compared periodically with thatdetermined by the ultraviolet absorptionphotometric method (10).

After the orientation session, subjectsinitiated daily urine collection upon thefirst day of menses of a new cycle. Subjectsplaced urine samples (3 ml) in a homefreezer after collection and brought themto the laboratory freezer upon the comple-tion of a full cycle. The urinary metabo-lites of the two major ovarian steroid hor-

mones, estrogen and progesterone, wereassessed by enzyme immunoassay (EIA) atthe Institute of Toxicologic and Environ-mental Health Research, University ofCalifornia, Davis, to monitor ovarian func-tion. The competitive, microtiter platesolid-phase EIA procedure for the mea-surement of ElConj and PdG is describedin detail by Munro and colleagues (11).Urinary hormone concentrations areexpressed as nanogram (ElC) or micro-gram (PdG) per milligram creatinine.

FVC, FEV1, and FEF25-75 preexposurevalues were subtracted from their respectivepost-exposure values and then divided bythe preexposure values to obtain percentchanges representing the treatment effectfor each protocol. We analyzed all data viaa two-way analysis of variance (ANOVA)with repeated measures (12), which testedfor gas concentration effects (filtered airversus 03 comparison), menstrual phaseeffects (F versus ML), and the interactionbetween menstrual phase and gas concen-tration effects. Upon obtaining a signifi-cant F ratio for main effects as the result ofeither gas concentration, menstrual phase,or their interaction, a paired-t post-hoc testwith modified Bonferroni correction (13)was applied to determine which particularmean values were significantly differentfrom others. Statistical significance wasaccepted at the p <0.05 level.

Group mean preexposure and post-exposure data for pulmonary function para-meters are presented in Table 2. Specificsignificant mean differences obtained byANOVA and post hoc procedures are alsoincluded. Post hoc analysis revealed signifi-cant gas concentration effects (filtered airversus 0 ) for FVC, FEV1, and FEF2575and significant gas concentration and men-strual phase interaction effects for FEV1and FEF25575 The group mean percentchanges for FEVI for each experimentalprotocol are illustrated in Figure 1. TheFEVI decrement of -18.1% observed in theF phase was significantly greater than the-13.1% mean value for the ML phase.This increased effect, which was not ob-served even as a trend for the filtered airexposures (Fig. 1), demonstrates the inter-action of03 and cycle phase.

Whereas the present data are not suffi-cient to define the cellular mechanismsinvolved, they suggest that ovarian steroids(particularly progesterone) may act tomodulate 03-induced inflammationoccurring in lung tissue. This associationis derived from the observation that thesensitivity of the lung to ambient 03 isvariable within the same woman andappears to depend on the phases of hermenstrual cycle. Although most of thesubjects exhibited sensitivity to 03 in bothphases, in general, normally menstruatingwomen appeared to be more sensitive tothe adverse effects of 03 during the Fphase (low progesterone) compared to theML phase (high progesterone) of theircycle.

All menstrual cycles used in the dataanalysis were characterized by low, unvary-ing PdG concentrations in the F phase,which were 0.9 ± 0.4 and 1.2 ± 1.4 jig/mgcreatine on the days of the F phase for airand 03 exposures, respectively. In con-trast, the PdG concentrations rose 1-2days after a pronounced mid-cycle ElCpeak and then declined before the nextmenstrual period 10-12 days later. ThePdG values on the day of the ML phasefiltered air and 03 protocols (8.6 ± 5.7and 8.5 ± 6.7 pg/mg creatine, respectively)were significantly elevated (p<0.0001)over F-phase levels. There were no signifi-cant differences between the treatmentgroups in mean cycle length, F-phaselength, luteal phase length, or urinary hor-mone concentrations.

Acute 03 exposure has been shown toresult in the production of inflammatorymediators (prostaglandins and thrombox-ane) in the lungs, airways, and plasma ofboth animals and humans (2,3,14). Avail-able data indicate that 03-inducedchanges in pulmonary function are theresult of prostaglandin stimulation ofneural afferents in exposed airways (15,16).In the present study, decreases in FEV1and FEF25-75 varied significantly withrespect to menstrual cycle phases, whichsuggests that there might be some fluctua-tion in the release or inhibition of pro-staglandins during the F and ML phases ofthe menstrual cycle.

Table 2. Group pre- and post-exposure values for pulmonary function

Filtered air 0.30 03F (1) ML (2) F (3) ML (4)

Parameter Pre Post Pre Post Pre Post Pre Post Specific mean differencesFVC (1) 3.91 ± 0.40 3.93 ±0.44 4.01 ± 0.45 4.00 ± 0.43 3.90 ±0.53 3.44 ± 0.72 3.99 ± 0.44 3.56 ± 0.58 11-3, 1-4, 2-3, 2-4FEVJ (1) 3.36 ± 0.41 3.40 ±0.44 3.45 ±0.46 3.51 ± 0.41 3.41 ± 0.50 2.82 ± 0.67 3.42 ± 0.48 2.99 ± 0.55 1-3, 1-4, 2-3, 2-4, 3-4

FEF27, (1/sec) 3.90 ±0.93 3.97 ±0.96 3.99 ±11.0 4.10 ±0.86 4.08 ±0.93 3.14 ±0.96 3.94 ±1.0 3.32 ±0.99 1-3,11-4, 2-3,2-4,3-4Abbreviations: F, follicular phase; ML, mid-luteal phase; FVC, forced vital capacity; FEV1, forced expiratory volume in 1 sec; FEF 25-7, forced expiratory flowbetween 25 and 75% of FVC. Values are means ± SD. Numbers in parentheses represent protocol number. Percent change values for each protocol [(post-pre)/(pre) x 1001 were used for statistical comparison to obtain specific mean differences (all significant at p<0.05).

Volume 101, Number 3, August 1993 243

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5

za..:.Ma

iDP.W.... Ig.............A-eFom5

-2C

C: .doiSStt;3 d,.......o,,i;.:;Sw.d'-'@FilSered.Air.0.30...._*

;:0);@:::oo Wg6Re!d::M RzcWia.:::b@eoiEbiaRee RSmR::ii e: PFd!RRRfoSd 11

m~ ~ ~ Flee Ai 0_0 _pm0

Figure. 1. Group mean percent change in forced expiratory volume in 1 sec (FEV,) on exposure to filteredair and to 0.30 ppm 03 in the follicular (F) and mid-luteal (ML) menstrual phases. The mean differencebetween the two 0.30-ppm 03 exposures was statistically significant (p< 0.05).

In normally menstruating females,gonadal steroid hormones are produced atvarying rates during the menstrual cycle.The F phase is characterized by baselineprogesterone concentrations and increasinglevels of estrogen up until ovulation. Afterovulation, the luteal or secretory phase ischaracterized by increasing levels of proges-terone. Progesterone secretion from thecorpus luteum reaches a peak approximate-ly 8 days after ovulation and is maintainedfor several days (17). Estrogen declinesdramatically after ovulation and then againrises to moderate levels during the MLphase. Both progesterone and estrogenlevels decrease at the end of a cycle, thustriggering menstruation (17).

It has been demonstrated in numerousstudies that sex steroids, essentially estro-gens and progesterone, exert a significanteffect on prostaglandin synthesis in varioustissues (7,8,18-20). In most cases, estradi-ol has been found to enhance the synthesisof prostaglandins E2 and F2a1 whereasprogesterone inhibits their secretion. Kellyand Smith (8) found that progesteronereduces prostaglandin F production by93-96% in human proliferative-phaseendometrium cultured for 2 to 3 days.

The increased FEV1 and FEF25-75 03responsiveness during the F phase could bethe result of progesterone interaction with03-induced prostaglandin synthesis. Pro-gesterone is evident in circulating serumand plasma samples in concentrations pro-portional to those observed in uterine tis-sues (11) and therefore can be found inrespiratory blood flow. During the Fphase of the menstrual cycle, when proges-terone is low, there may be no inhibitionof prostaglandin synthesis after 03 expo-sure. In contrast, with higher concentra-tions of progesterone during the ML

phase, there may be some inhibition ofprostaglandin activity associated with aconcomitant decrease in inflammation,which results in slightly smaller decrementsin FEV1. Although the involvement ofprogesterone in modulating 03-inducedproduction of prostaglandins is only sug-gestive, it is the only cyclic hormone thatfits the profile required to have this effect(i.e., high in the ML and low in the Fphases). However, additional investigationis warranted in which the role of proges-terone (and other hormones) can be direct-ly evaluated, for example, by adding prog-esterone to an exposed system.

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2. Fabbri LM, Aizawa H, Alpert SE, Walters EH,O'Byrne PM, Gold BP, Nadel JA, HoltzmanMJ. Airway hyperresponsiveness and changes incell counts in bronchoalveolar lavage after ozoneexposure in dogs. Am Rev Respir Dis 129:288-291(1984).

3. Seltzer J, Bigby BG, Stulbarg M, Holtzman MJ,Nadel JA, Ueki IF, Leikauf GD, Goetzl EJ,Boushey HA. 03-induced change in bronchialreactivity to methacholine and airway inflamma-tion in humans. J Appl Physiol 60:1321-1326(1986).

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8. Kelly RW, Smith SK. Glucorticoids do notshare with progesterone the potent inhibitoryaction on prostaglandin synthesis in humanproliferative phase endometrium. Prosta-glandins 33:919-929(1987).

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11. Munro CJ, Stabenfeldt GH, Cragun JR,Addiego LA, Overstreet JW, Lasley BL. Re-lationship of serum estradiol and progesteroneconcentrations to the excretion profiles of theirmajor urinary metabolites as measured byenzyme immunoassay and radioimmunoassay.Clin Chem 37:838-844(1991).

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13. Keppel G. In: Design and analysis: a re-searchers handbook. Englewood Cliffs, NewJersey:Prentice Hall, 1991;169-170.

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15. Lee L-Y, Dumont C, Djokic TD, Menzel TE,Nadel JA. Mechanism of rapid shallow breath-ing after ozone exposure in conscious dogs. JAppl Physiol 46:1108-1109(1979).

16. Hazucha M, Baks D, Bromberg P. Mechanismof action of ozone on the human lung (ab-stract). Am Rev Respir Dis 133 A214(1986).

17. Franz WB. Basic review: endocrinology of thenormal menstrual cycle. Primary Care 15:607-616(1988).

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19. Abel MH, Baird DT. The effect of 17b-estra-diol and progesterone on prostaglandin pro-duction by human endometrium maintained inorgan culture. Endocrinology 106:1599-1606(1980).

20. Schatz F, Markiewicz, Gurpide E. Effects ofestriol on PGF2a output by cultures of humanendometrium and endometrial cells. J SteroidBiochem 20:999-1003(1984).

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