Vol. 2, 221- 228. May/June 1 99 1 Cancer Epidemiology, Biomarkers & Prevention 223

Detection of Mutagens in Cervical Mucusin Smokers and Nonsmokers1

Elizabeth A. Holly,2 Rosemary D. Cress, David K. Ahn,Diana A. Aston, Jennifer J. Kristiansen, Rebekah Wu,and James S. FeltonDepartnie’nt of Epmde’mmology and Biostatistics, University of CaliforniaSan Francisco School of Medicine, San Francisco 94109 [E. A. H.,R. I). C., D. K. A., D. A. A., I. I. K.], and Biomedical Sciences Division,Lawre’nce Livermore National Laboratory, Livermore’ 94550�9900 [R. W., I. S. F.], California

Abstract

The Salmonella mutagenicity test was used to analyzecervical mucus specimens from 364 smokers and 333nonsmokers to determine whether the associationbetween smoking and mutagenic cervical mucus thatwe reported previously among women diagnosed withdysplasia would apply to a larger group of healthywomen (E. A. Holly et al., J. Natl. Cancer Inst., 76: 983-986, 1986). Women smokers and nonsmokers betweenthe ages of 18 and 49 who attended eleven clinics andphysicians’ offices in the San Francisco Bay area for aroutine Pap smear were examined to determinewhether smokers were more likely to have mutagenicsubstances in their cervical mucus. About 4% ofsmokers and 8% of nonsmokers had positivemutagenicity test results (P 0.02). Cervical mucuswith a large number of microorganisms was morelikely to have a positive mutagenicity test result thanthat with fewer microorganisms (test for trend, P =

0.01). Mutagenicity results varied by race and cliniclocation but were not associated with smokingbehavior, sexual behavior, gynecological diagnosis, ordiet. Further work is needed to develop methods todetect mutagens in specific body fluids.

Introduction

Mutagens are substances that have a genotoxic effect onhuman tissues, and the presence of mutagens in body

fluids is an indicator of the ingestion, excretion, andpossible metabolism of these chemicals (1). !n vitro de-tection of mutagens in body fluids was made possible inthe 1970s by the development of the Sa!mone!!a/mam-malian-microsome mutagenicity test (hereafter called theSa!mone!!a mutagenicity test) (2). Mutagenic activity wasdemonstrated in cigarette smoke condensates (3) and in

Ree’ive’d 9/23/92.1 Supported by National Cancer Institute Research Grant CA42440 from

the NIH.2 To whom requests for reprints should be addressed, at Department of

Epide’miology and Biostatistics. University of California San FranciscoSchool of Medicine, 1 388 Sutter Street, Suite 920, San Francisco, CA

94109.

concentrated urine from smokers by Yamasaki and Ames(4). The association of mutagenic urine with smoking andpassive smoking has been confirmed by several recentlyreviewed studies (5) and has provided a possible expla-nation for the higher risk of bladder cancer among smok-ems. Mutagenicity testing of other body fluids includinggastric juice (6), amniotic fluid (7), and sputum (8) hasbeen attempted less frequently and has met with meth-odologic problems and yielded inconsistent results.

Women smokers have been noted to have an ele-vated risk of cervical cancer compared to nonsmokersafter controlling for other confounding factors, but thebiological rationale for the association has not been es-tablished (9, 10). Cotinine and nicotine have been de-tected in the cervical mucus of smokers (11) and incervical lavage from women exposed to passive smoke(1 2), supplying evidence that constituents of smoke reachthe uterine cervix and providing a valuable indicator ofsmoking status (1 3). Cotinine and nicotine are not amongthe mutagenic components of tobacco smoke (14), al-though they may be a marker for the presence of anundetected mutagen (13). In 1986, we reported an as-sociation between cigarette smoking and mutagenic cen-vical mucus in women with cervical dysplasia (15). Thepurpose of the current study was to determine whetherthe association between smoking and mutagenic cervicalmucus that we found in women who had been examinedat the UCSF3 Dysplasia Clinic and who were at high riskof cervical cancer (15) would apply to a larger group ofhealthy women smokers and nonsmokers.

Materials and MethodsThe current study was conducted in women who at-

tended 1 1 San Francisco Bay area physicians offices andclinics, including seven Planned Parenthood clinics, be-tween May 1987 and November 1990. Women smokersand nonsmokers between the ages of 18 and 49 wereasked to participate in the study when they visited theirphysicians for a routine Pap smear. In addition to the 631normal, healthy women recruited from the physiciansoffices, 66 women from the UCSF Dysplasia Clinic wereasked to participate to determine whether smokers withdysplasia had different results on the test for mutagenicityof cervical fluids than did healthy women smokers andwomen who did not smoke.

Women who consented to participate were inter-viewed by professional interviewers about smoking hab-its, passive smoking environment, sexual and reproduc-

3 The abbreviations used are: UCSF, University of California San Fran-cisco; LLNL, Lawrence Livermore National Laboratory; DMSO, dimethyl

sulfoxide; OR, odds ratio; CI, confidence interval.

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224 Mutagens in Cervical Mucus

tive history contraceptive use, history of sexually trans-mitted diseases, and recent diet. The diet questions werelimited to foods and drinks thought to be related tomutagenicity of body fluids and included questions about

consumption of alcohol; soft drinks; coffee; crucifemousvegetables; and grilled, broiled, and barbecued meats.

Smokers and nonsmokers were recruited from eachclinic in approximately equal numbers. Former smokers(n = 32) had not smoked for a mean of 8 years (SD =

6.4) and were included with nonsmokers. Current smok-ems were asked about number of years smoked, usualnumber of cigarettes per day, and time of day they lastsmoked a cigarette.

Cervical mucus specimens were collected by phy-sicians after the routine Pap smear was obtained. Thewoman’s cervix was flushed and neflushed with 1 .5 ml ofsterile nonpynogenic electrolyte solution, and the fluidcontaining cervical mucus was retrieved, placed in a testtube, and frozen. All specimens were kept frozen untilthey were transferred to LLNL for testing. Upon arrival at

LLNL, the cervical mucus specimens were immediatelycultured by laboratory personnel for yeast, lactobacillus,

and other microorganisms. Microorganisms other thanlactobacilli were cultured on TSA agar, and yeast andlactobacilli were grown using selective agams, Sabourauddextrose and Rogosa SL agars, respectively. Cervical mu-cus specimens were kept frozen (-20#{176}C) at LLNL for nolonger than 14 days, and Sa!mone!!a mutagenicity testswere completed in batches after adequate specimenswere collected from all medical clinics. Sa!mone!!a mu-tagenicity tests were conducted according to the plateincorporation method described by Ames and Maron(16).

The Sa!mone!!a test for mutagenicity uses a strain ofSa!mone!!a typhimurium bacteria that contains a selectedmutation to prevent the bacteria from synthesizing theamino acid histidine. Certain mutagens will cause a DNAframeshift change that will reverse this mutation andallow the bacteria to grow and divide, and an increasednumber of neventant bacteria over background providesan indicator of the presence of a mutagen. Theme areseveral strains of the specially developed Sa!mone!!a

bacteria, and each contains a different mutation for thegene that controls histidine metabolism, allowing me-sponse to different types of mutational events.

In the current study, cervical mucus that had beendissolved in the organic solvent DMSO was combinedwith molten soft agam; TA98 Sa!mone!!a bacteria, thestrain most sensitive to mutagenic dietary factors and totobacco products such as polycyclic aromatic hydnocan-bons (PAHs) and aromatic amines; and 59, a metabolicagent prepared from livers of Anoclor-treated mats thatpermits detection of substances that must be metabo-lized to be mutagenic (i7). The cervical mucus of 56subjects was tested with TAiOO, another Sa!mone!!astrain sensitive to cigarette smoke constituents. The testresults for subjects tested using TA1 00 did not differ from

those tested with TA98, and the results were combinedfor analysis. The specimens were tested in duplicate attwo doses: 200 and 400 ,�l of cervical secretion, thehighest doses compatible with the test and the availabilityof sample. Routine examination of the background lawnof bacterial growth revealed no toxicity at either the 200-or the 400-gil dose. After the molten agam cooled andsolidified, the plates were inverted and incubated at

37#{176}Cfor 48 h. After incubation, the number of nevertantcolonies was counted and reported as number of multi-pies times the number of spontaneous reventant colonies.The test was considered positive if the number of never-(ant colonies was 2.0 times the background count. Apositive control that contained 2-aminoanthracene anda negative control that contained DMSO and 59 but notest substance were used with each batch. Laboratorypersonnel had fl() knowledge of the smoking status ofthe subjects, of responses to questions asked in theinterview, on of Pap smear results. The same technicianconducted all tests for mutagenicity.

The above methodology is standard (16) and wasused in both this and our earlier study (15), althoughdifferent laboratories conducted the tests. However, amodification of the sample preparation was made in thecurrent study by LLNL personnel to reduce the possibilityof contamination by bacteria normally found in cervical

mucus. The cervical samples were thawed, and an aliquotwas plated for the culture of microorganisms. Prior tomutagenicity testing, the cervical mucus was dissolved in

DMSO at a ratio of 1:1 DMSO to cervical sample andallowed to incubate overnight (18 h) at 25#{176}Cto sterilizethe samples before testing. In the earlier study (15), thespecimens were incubated with the DMSO for 30 to 60mm prior to plating, which was standard sample prepa-ration at the time the tests were conducted.

The samples obtained from the women attendingthe UCSF Dysplasia Clinic were split to evaluate theeffect of the modified sample preparation on test results.One-half of each sample was tested using the abovemethod, which included overnight incubation withDMSO. The other half of the sample was tested usingthe sample preparation used in the earlier study in whichcervical mucus specimens were incubated with DMSOfor 30 to 60 mm prior to combining with the otheringredients.

Data analysis was conducted using BMDP statisticalsoftware programs (18). Univaniate and multivaniate oddsratios were computed for factors of interest among smok-ers and nonsmokers using logistic regression analysis,with 95% confidence intervals, P values, and tests fortrend computed when appropriate (19).

Results

Eighty-five % of women who were approached for thisstudy agreed to participate, with 364 smokers and 333nonsmokers interviewed and examined. Sixty-eight % ofthe women who smoked reported that they smoked lessthan half a pack of cigarettes a day and were classifiedin some analyses as light smokers. Women who smokedmore than half a pack a day were classified in these sameanalyses as heavy smokers.

Table 1 shows the test results for women by smokingstatus, race, and clinic. About 4% of smokers and 8% ofnonsmokers had a positive result on the Sa!rnone!!a testfor mutagenicity (P = 0.02). A somewhat higher propon-tion of nonsmokers than smokers had a positive test inall race groups except Hispanics, although the resultscould have been due to chance. Asian and Hispanicwomen were somewhat more likely to have a positive

test than white non-Hispanic and black women, but theresults could have been due to chance. A higher propor-(ion of nonsmokers than smokers had a positive test

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Cancer Epidemiology, Biomarkers & Prevention 225

Table I Number and percentage positive on the Salmonella testfor mutagenicity among women smokers and nonsmokers

by race and clinic

Smokers NonsmokersTotal )n = 364) (n = 333)

NN % positive N �/c positive

Total 697 13 3.6 26 7.8’

Race

White, non-Hispanic 427 7 3.1 i3 6.4White’, Hispanic 50 2 1 i.i 3 9.4Black 170 1 1.0 6 8.2

Asian 30 1 6.7 3 20.0

ClinicPA 222 4 3.5 5 4.7

UCS 64 1 3.8 2 5.3

PP 276 6 4.1 14 10.8’

EO 114 2 2.9 5 10.9FR 8 0 0.0 0 0.0

.,I) = 0.02 for difference between smokers and nonsmokers.

I’ Dvsplasia Clinic.

‘ P=0.05.

result in all clinics except Clinic FR, where none of thewomen had positive test results. However, the differencebetween smokers and nonsmokers was statistically sig-nificant only for women who attended Clinic PP (P =

0.05), where the number of positive test results washighest.

Table 2 displays the results of the Sa!mone!!a muta-

genicity test for selected dietary factors. Women whohad eaten meat or crucifemous vegetables in the 24 hprior to the test had similar results on the test for muta-genicity to those who had not eaten these foods. Womenwho had consumed broiled meat were slightly morelikely to have mutagenic cervical mucus, and this resultapproached statistical significance (OR = 2.3; CI = 0.89-5.8; P = 0.09).

Table 3 shows the results of the Sa!mone!!a test formutagenicity by number of cervical microorganisms andsample preparation method. Five % of samples testedwith the original sample preparation and 6% of samplestested with the modified sample preparation had a pos-itive test result. When the modified sample preparationmethod was used, cervical mucus with a large numberof cervical microorganisms was more likely to have apositive Sa!mone!!a mutagenicity test result than that withfewer microorganisms. Although the OR was elevated toabout 1 .5 for the number of microorganisms greaten than200 microorganisms/mI using the original sample prepa-ration method, the number of positive tests was very

small.Women who were currently using oral contracep-

tives and those who were not were equally likely to havea positive test for mutagenicity (OR = 0.87; Cl = 0.41-1 .8; P = 0.84). When compared with women who lasthad sexual intercourse in the 2 days before the exami-nation, women who last had intercourse more than 5

days before the examination were somewhat more likelyto have mutagenic mucus (OR = 2.7; Cl = 0.88-8.2; P =

0.09).Women who had a gynecological diagnosis of cen-

vical dysplasia on the day of specimen collection (n =

54) were as likely as women who did not have a diagnosis

Table 2 Number and percentage positive on the Salmonella testfor mutagenicity by selected food consumption

in the 24 h prior to sample collection

Yes NoFoods consumed

N % positive N % positive

Cruciferous vegetables 13 7 26 5

MeatBeef 19 6 20 5

Pork 9 5 30 6Poultry 13 5 26 6

Meat preparationBroiled 7 11’ 32 5

Fried 13 5 26 6

Barbecue 4 7 35 6Grilled 3 7 36 6Baked 12 6 27 6

Boiled 4 5 35 6

‘ P = 0.09.

of dysplasia to have had a positive result on the Sa!mo-ne!!a test for mutagenicity. Nine of these women (17%)were being followed at the UCSF Dysplasia Clinic, while44 were diagnosed on routine Pap smear at the otherclinics. When we combined women from all clinics, 7%of women with dysplasia had a positive test result com-pared to 6% of women who did not have a diagnosis ofdysplasia (OR = 1.4; Cl = 0.14-4.3; P = 0.79).

Neither time since last cigarette non number of cig-

amettes per day were related to results on the test formutagenicity. Women who had smoked within 2 h be-fore the sample collection had mutagenicity test resultssimilar to those of women who had last smoked morethan 2 h previously (OR = 1 .9; Cl = 0.54-6.3; P = 0.42).Women who were classified as heavy smokers were nomore likely to have a positive result on the test formutagenicity than were women who were classified aslight smokers (OR = 0.64; Cl = 0.06-2.6; P = 0.71).

Discussion

In contrast to our previous study in women with cervicaldysplasia (1 5), the test for mutagenicity of cervical mucusin the current study detected no difference betweenhealthy women smokers and nonsmokers. Differenceswere noted between smokers and nonsmokers in thecurrent study on several other factors, and these havebeen published previously (20). Smokers were slightlyolden than nonsmokers (mean age, 29.7 years versus 28.3years) (20), and subsequent analyses were adjusted forage. Smokers were less educated, less likely to be His-panic, and somewhat more likely to be black whencompared to white non-Hispanic women. They weremore likely to be separated, divorced, or living as ifmarried in a relationship of at least 6 months whencompared to married and single women, and consumedlarger quantities of coffee, beer, liquor, soft drinks, and

beef than did nonsmokers (20). Smokers also were moresexually active with earlier age at first intercourse and ahigher number of sexual partners, and smokers had ahigher number of cervical microorganisms (20).

Interpretation of the Sa!mone!!a mutagenicity testresults in the current study is hampered by the lownumber of positive results. The different results from the

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226 Mutagens in Cervical Mucus

Table I Odds ratios and 9S�/c confidence’ intervals for results of the’ Salmone’Ila te’st for mutage’nicitv bs s.miiiple 1)re’P.mr,mtmimn methodand ce’rvic al culture’ results for smokers and nonsmokers coml)ine’d

Colony counts/mI

cervical wash

Modified sam pIe preparation’ Original s,iiii pIe’ pre’3).ir,Ition”

Positive’

test results

Negative’

test results OR 95% CI PPositive’

test results

Negative’

te’st results OR 95% (l I’

( N = 36) ( N = 588) )N= 3 ( ( N=6 1

Microorganisms”

�200 5 169 1.0 1 26 1.0

201-8500 9 194 1.6 0.47-5.5 0.6(1

>8500 22 225 3.3 1.2-10.2 0.02 2’ 35 iS 0.11 17.3 (176

Test for trend 0.Oi

‘See text for definition.S In ludes yeast, other microorganisms, not lactobacillus.

, 201 to >8500.

two studies may be attributable to different study popu-lations on to a change in the test methodology. Thepopulation in our earlier study (1 5) comprised 82 womenfrom the UCSF Dysplasia Clinic, while the population inthe current study included 631 women from other Bayarea clinics and 66 women from the UCSF DysplasiaClinic. The current study also used different methods toprepare the mucus specimens for testing by incubatingthe specimens with DMSO overnight to prevent contam-ination by normal cervical microorganisms. However,neither sample preparation method nor dysplasia statusaffected the proportion of positive test results.

In our earlier study, women who had smoked acigarette within 7 h of the sample collection were morelikely to have a positive test result for mutagenicity thansmokers who had not consumed a cigarette in that timeperiod (iS), but neither time since last cigarette nornumber of cigarettes was associated with mutagenicityin the current study. Both number of cigarettes and time

since last cigarette have been shown to be associatedwith mutagenic urine (4, 21).

With the possible exception of broiled meat, a pos-itive result on the test also was not associated with theingestion of foods that have produced detectable muta-gens in urine in other studies (22, 23). Cooked meatscontain heterocyclic amine carcinogens (24), and thisstudy noted a higher consumption of some meats amongsmokers (20). Dietary mutagens may not be excreted incervical mucus on the test may have been unable todetect the levels of mutagens that were present.

Results from our earlier study (15) noted a higherproportion of mutagenic mucus among women smokerswho attended the UCSF Dysplasia Clinic, raising the

question of whether these results were related to thediagnosis of dysplasia. A study in India of 98 women,most of whom were nonsmokers, noted that 23.4% ofwomen with cervical intmaepithelial neoplasia had muta-genic cervical mucus compared to 3.9% of healthy con-trols (25, 26). In our current study healthy women andwomen with dysplasia had similar results on the test formutagenicity, but the number of women with dysplasiawas too small to detect a difference between the smokersand the nonsmokers.

The sample preparation methodology used in thisstudy is the same as that used in a small study of 55healthy women in Washington, DC (13, 27). That studynoted that 9% of smokers and 1 3% of nonsmokers had

mutagenic cervical mucus when the mucus was incu-bated overnight with DMSO before testing. This result iscomparable to the 4% of smokers and 8% of nonsmokerswho had a positive test in the current study using thesame sample preparation methods. The laboratory per-sonnel in the earlier study (27) noted the presence ofatypical colonies in cervical samples not incubated over-night with DMSO and observed that these colonies werefound more frequently in cervical mucus from smokersthan that from nonsmokers. The bacterial contaminationwas identified as Staphy!ococcus, Streptococcus, diphthe-roid, and micnococcus species and non-Sa!rnone!!a

Cram-negative rods (13). The current study also noted ahigher number of microorganisms in the cervical mucusof smokers (20). In the current study, mucus specimenswere plated for culture of microorganisms prior to incu-bation with DMSO, and cervical mucus with a highnumber of cervical microorganisms was more likely tohave a positive test for mutagenicity. It may be that evenovernight incubation with DMSO failed to kill all contam-ination, and other methods to eliminate normal cervicalmicroorganisms need to be developed. Nioshoka et a!.used ampicillin to inactivate normal flora in saliva (28),and Parashani et a!. used ampicillin as well as overnightincubation with DMSO to sterilize cervical mucus (26).

Little has been published on the methods requiredto isolate mutagens from body fluids other than urine.Urine is relatively easy to obtain in large quantities andto analyze. The methods used to isolate mutagens fromurine have been developed and refined over a numberof years and are now well documented (2, 4, 16, 21, 29,30). Other body fluids such as cervical mucus, saliva,breast fluid, and gastric juice are somewhat more difficult

to obtain in quantity, and the methods required to detectmutagens in these fluids are not well defined. Unlikeurine, these biological fluids tend to be complex mixturesthat contain cells, proteins, enzymes, and other chemi-cals (6, 28) that may interact in unpredictable ways withthe bacterial test system for mutagenicity. Human salivahas been noted to inactivate the mutagenicity of thecondensate of cigarette smoke, and this effect is thoughtto be caused by biochemical reactions with enzymes orby biophysical adsorption by mucus on bacteria on otherhigh-molecular-weight constituents (28).

Expectorates, composed of sputum and mucus, fromworkers in an aluminum factory were found to be mu-tagenic after extraction of adsorbed mutagens with cy-

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Cancer Epidemiology, Biomarkers & Prevention 227

clohexane (8). However, control subjects who smoked

but were not exposed to factory aim did not have muta-genic expectorate (8). The detection of mutagens in

gastric juice has been shown to be affected by thepresence of histidine, which increased the number of

mevertant colonies, and by the presence of substances

that inhibit mutagenesis (6). Detection of mutagens inconcentrated (freeze-dried) amniotic fluid of smokerswas hampered by methodological problems and yielded

inconsistent results, possibly because of the presence of

histidine in amniotic fluid (7). Further work is needed to

develop methods to isolate mutagens from cervical mu-

cus. Mucus may contain substances similar to those in

saliva that inhibit mutagenic activity. Mutagens in cervicalmucus may need further extraction, such as that used on

expectorate, to remove them from extraneous

constituents.Researchers using the Sa!mone!!a mutagenicity test

have noted that minor changes in the procedure cancause different results. DeRaat et a!. (31) documented

that a change as minor as differing quantities of bottom

agan on the incubation plates changed the numbers ofspontaneous mevertants in the Sa!mone!!a mutagenicity

test using TA98 and other strains of Sa!mone!Ia. Increas-ing the number of TA98 bacteria added to the test has

been noted to increase the sensitivity of the test to

benzo(a)pyrene in the urine of smokers (30). The effectof incubating cervical mucus overnight with DMSO has

not been published. A recent analysis of the stability of

mutagens in the frozen urine of smokers reported that

mutant response decreased slightly with increased stor-

age time (up to 1 75 days), although the results were not

statistically significant (32). Storage time in the currentstudy was limited to 14 days. While DMSO is generallythe solvent of choice for mutagenicity assays because of

its ability to dissolve a variety of chemical substances, itis not always the best solvent. It is known to react with

certain chemicals and to inhibit the mutagenic effect of

some chemicals (33). Although it is unlikely that DMSOwas able to inactivate the mutagens in the cervical mu-

cus, it is not currently known how DMSO reacts with the

various components of cervical mucus, especially in the

prolonged period of time used in the current study.However, positive control specimens did remain positive

and were not significantly affected by the overnight in-

cubation with DMSO.Men and women are exposed to a variety of muta-

genic chemicals in the environment and workplace. Sinceits development nearly 20 years ago, the Sa!mone!!a test

for mutagenicity has demonstrated its value to detectingthese mutagens and their metabolites in human bodyfluids, especially urine. While the methods to isolate

mutagens from urine have been improved during this

period, the methods used to isolate mutagens from other

body fluids are less well understood, and further work is

needed.

Acknowledgments

The authors are grateful to the physicians at the 1 1 clinics in northern

California who contributed their effort to this study through specimencollection and subject recruitment.

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1993;2:223-228. Cancer Epidemiol Biomarkers Prev   E A Holly, R D Cress, D K Ahn, et al.   nonsmokers.Detection of mutagens in cervical mucus in smokers and

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