age-related negative associations between parameters of ... · i this study was supported by grant...

Vol. 6, 943-948, November 1997 Cancer Epidemiology, Biomarkers & Prevention 943

Age-related Negative Associations between Parameters of Cytogenetic

Damage and ex Vivo (±)-anti-Benzo(a)pyrene Diolepoxide-induced

Unscheduled DNA Synthesis in Smoking l

R. H. Stierum,2 G. J. Hageman, M. H. M. van Herwijnen,M. S. E. van der Veer, and J. C. S. Kbeinjans

Department of Health Risk Analysis and Toxicology. University of Limburg,

6200 MD Maastricht, The Netherlands

Abstract

Chemical or physical modification of DNA may cause anincrease in genomic mutations or other geneticalterations, which may ultimately result in the onset ofcancer. To avoid these deleterious effects of DNAdamage, humans possess DNA repair mechanisms.Decreased DNA repair, induced ex vivo by UV light orionizing radiation in human peripheral bloodlymphocytes (PBLs), has been associated with aging. Theaim of this study was to investigate whether repair ofDNA damage, after ex vivo exposure of PBLs obtainedfrom smokers (n = 20) to (±)-anti-benzo(a)pyrenediolepoxide [(±)-anti-BPDE], which is a mixture ofreactive metabolites from the environmental carcinogenbenzo(a)pyrene, is also associated with age. Furthermore,age-related associations between ex vivo (±)-anti-BPDE-induced DNA repair and the frequency of endogenouscytogenetic damage (sister chromatid exchangefrequencies and micronuclei frequencies) in PBLs wereevaluated. A statistically significant negative associationwas observed between ex vivo (±)-anti-BPDE-inducedunscheduled DNA synthesis and age of the donors. Also,parameters of endogenous bymphocytic cytogeneticdamage were negatively associated with ex vivo (±)-anti-

BPDE-induced unscheduled DNA synthesis and positivelyassociated with age in this population. It is concludedthat, with increasing age, a decrease in lymphocyticexcision repair capacity may be responsible for increasedlymphocytic DNA damage in smokers.

Introduction

Humans are continuously exposed to various agents that may

chemically or physically modify cellular DNA. It is believedthat such modifications result in a time-dependent accumuba-

tion of genomic mutations or other genetic alterations ( 1 . 2). Ifthese alterations take place in proto-oncogenes or tumor sup-pressor genes. cell cycle control may be lost, ultimately result-

ing in the onset of cancer. To protect cellular DNA from thesedeleterious effects of DNA damage, prokaryotic and eukaryotic

cells, including human cells. possess DNA repair mechanisms.The importance of adequate DNA repair mechanisms in pre-venting carcinogenesis is exemplified by the DNA repair-defective, autosomal recessive disease xeroderma pigmento-sum. Cells obtained from individuals having this disease aredeficient in nucleotide excision repair of barge DNA adducts.

for example, UV-induced cycbobutane pyrimidine dimers.Moreover, these individuals develop skin cancer. even at earlyages. and have a 2000-fold increased risk of cancer compared

to normal individuals (3).

Recently, decreased UV-induced DNA repair capacity ofPBLs3 was associated with development of sunlight-inducedskin cancer in the normal human population (4). Furthermore,

this decrease in DNA repair capacity was negatively associatedwith age. Other studies also report negative associations be-

tween the extent of DNA repair. induced by various DNA-damaging agents cx viva in PBLs, and age of the donor (5-7).Thus, it can be hypothesized that the ability to perform ade-quate DNA repair declines with increasing age. In turn, reducedrepair capacity would result in increased persistence of DNA

damage and increased cancer risk, particularly in populationsexposed to environmental carcinogens. At present. the poten-

tiabby age-related negative association between the capacity ofDNA repair mechanisms and DNA damage in vito has not yetbeen investigated in detail in those human populations that are

at risk for cancer development.In humans, cytogenetic damage has been positively asso-

ciated with smoking habits (8-1 1 ). Moreover, increased cyto-

genetic damage was found to be associated with increasedcancer risk ( 12). Therefore, it is interesting to hypothesizewhether, in smokers, an age-rebated decline in DNA repaircapacity increases steady-state levels of smoking-induced cy-

togenetic damage and, thereby, augments the risk for develop-ment of cancer in this carcinogen-exposed population with

increasing age.In a previous study,4 the effects of in vito nicotinic acid

supplementation on niacin state, poby(ADP-ribosyl)ation. cyto-

genetic damage, and DNA repair in smokers were described.

Received 4/27/95; revised 6/30/97: accepted 7/31/97.The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertise,ne,it inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

I This study was supported by Grant 28-2124 from the “Praeventiefonds” (The

Hague. The Netherlands).

2 To whom requests for reprints should be addressed, at Laboratory of Molecular

Genetics, NIH, National Institute on Aging, Gerontology Research Center, Box01, 4940 Eastern Avenue, Baltimore, MD 21224. Phone: (410) 558-8090; Fax:

(4 1 0) 558-8 157: E-mail: [email protected].

3 The abbreviations used are: PBL. peripheral blood lymphocyte: ( � )-ai:Ii-BP[)E,

( ± )-anti-benzo(a)pyrene diolepoxide: UDS. unscheduled DNA synthesis: HER.

base excision repair: NER. nucleotide excision repair: SCE. sister chromatid

exchange: MN, micronuclei: PHA. phytohemagglutinin.

4 R. H. Stierum, G. J. Hageman, M. H. M. van Herwijnen. M. S. F. van der veer.A. P. H. Vankan, and J. C. S. Kleinjans. Nicotinic acid supplementation: effects

on niacin status, DNA repair and cytogenetic parameters in lymphocytes of

smokers, submitted for publication.

on March 26, 2020. © 1997 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from

http://cebp.aacrjournals.org/

944 Age.related DNA Damage and Repair Changes in Smokers

Here, we report age-rebated associations between DNA repair

and DNA damage in this carcinogen-exposed population. Asparameter of DNA repair, (±)-anti-BPDE-induced UDS was

determined ex vivo in PBLs. (±)-anti-BPDE, a mixture ofmetabolites from the carcinogen benzo(a)pyrene, which is pres-

ent in cigarette smoke, induces a variety of DNA lesions thatare substrates for BER or NER mechanisms (13-15). These

repair mechanisms are expected to also act on cigarette smoke-induced DNA damage in vivo. To compare this estimate of

DNA repair capacity with the extent of in vivo (smoking-

induced) DNA damage, SCE frequencies and MN frequencieswere determined in untreated PBLs, after stimulation to prolif-

eration ex vivo.

Materials and Methods

All chemicals were purchased from Merck (Darmstadt, Ger-

many), unless otherwise specified. Cell culture media and sup-plements were obtained from Life Technologies, Inc., (Paisley,

Scotland). Twenty healthy male human smokers who smoked atleast 10 cigarettes per day were included in this study. The age

of the subjects ranged from 21 to 55 years. Mean (± SD) values

for number of cigarettes smoked per day, pack-years [pack ofcigarettes smoked per day X number of years of smoking (at 20

cigarettes/pack)J, and alcohol and coffee consumption were:

19.6 ± 5.8 cigarettes/day; 14.8 ± 9.8 pack-years; and 12.3 ±

9.3 glasses/week and 5.3 ± 2.6 cups/day, respectively. Allvolunteers agreed to participate to this study by giving their

written informed consent. The study protocol was approved by

the Medical Ethical Commission of the University of Limburg

(Maastricht, The Netherlands).Heparmnized venous blood for assaying cytogenetic dam-

age and DNA repair was collected from each donor on 2

different days, within a period of 8 weeks. Methods for deter-

mination of SCE an MN have been described elsewhere (16).Briefly, for determination of endogenous SCE levels, 0.4 ml of

blood were added to 5.0 ml of RPM! 1640, supplemented with

10% FCS, 100 pg/mb streptomycin, 100 units/mI penicillin, 5mM L-glutamine, and 50 units/mb heparmn (RPMI 1640 complete

medium). PHA (0.2 ml) was added (final concentration, 52

sg/mb), and cells were cultured for 24 h at 37#{176}C.Bromode-oxyuridine (Serva, Europe) was added (final concentration, 58

.LM), and cultures were incubated for another 48 h. One h prior

to harvesting, 100 �l of colcemid were added. Then cells werehypotonized in 75 mM KC1 for 10-15 mm and fixed withmethanol:acetic acid (3: 1 , v/v). Metaphase slides were prepared

and stained by means of the Hoechst-plus-Giemsa technique( 17). Before microscopic evaluation, the slides were encoded.

Slides were evaluated by a webb-trained observer, and SCEscores were checked by a second independent observer. From

each subject, for each time point, at least 20 metaphases,containing 40 chromosomes as a minimum, were analyzed for

SCEs.

Endogenous MN frequencies were determined using the

assay of Fenech and Morley (18). Venous heparmnized blood(0.4 ml) was cultured in 5 ml of RPMI 1640 complete medium

and 0.2 ml of PHA (final concentration, 52 �sg/ml) to stimulateT-cell proliferation. To block cytokinesis, 44 h after culturing,

cytochalasin B (Sigma, Axeb, The Netherlands) was added to afinal concentration of 6 �tg/mb. After an additional cultureperiod of 28 h, cultures were harvested as described above, and

slides were prepared and stained with 3% Giemsa for 20 mm.

Prior to microscopic analysis, slides were encoded. Per mdi-vidual, 1000 binucleated cells were analyzed for the presence of

Table I Means (±SD) and ranges of (±)-a,iti-BPDE-induced UDS, SCE

frequencies. and MN frequencies in PBLs obtained from male smokers

Parameter Mean ± SD Range

UDS (net grains/nucleus)

(±)-anti-BPDE-treated cultures 24.9 ± 6.5 14.0-34.7

DMSO-treated cultures I .81 ± 0.6 0.7-3.0

(±)-anti-BPDE-induced UDS 23.1 ± 6.2 12.2-32.5

SCEs/cell (nontreated cultures) 6. 1 ± 1 .0 4.7-8.1

MN/bOO binucleated cell (nontreated cultures) 5.3 ± 2.0 2.5-9.5

MN by a well-trained observer and checked by a second inde-pendent observer.

To estimate in vivo DNA repair capacity, (±)-anti-BPDE-induced UDS was determined in whole-blood cultures. (±)-

anti-BPDE [(±)-7�3,8a-dihydroxy-9a, lOce-epoxy-7,8,9, lO-tet-

rahydrobenzo(a)pyrene; Midwest Research Institute, KansasCity, MO] stock solutions were prepared in anhydrous DMSOand stored at -20#{176}Cin the dark prior to single use. (±)-anti-

BPDE (or DMSO for solvent control-treated cultures) was

added to a large volume of RPMI 1640, supplemented with10% FCS, 100 �g/ml streptomycin, 100 units/ml penicillin, 5

mM L-glutamrne, and 50 units/mb heparin, of which 2.5 ml weresubsequently added to 0.2 ml of blood, to a final concentration

of 1.0 �LM (final concentration of DMSO, 0.4%). Within 10 mm

after addition of the carcinogen, [methyb-3H]thymidine (Amer-sham; specific activity, -80 Ci/mmol) was added to a final

concentration of 10 pCi/mb. Twenty-four h after initiation ofthe cultures, cells were hypotonized in 75 msi KC1 for 10-15

mm and fixed with methanob:acetic acid (3: 1, v/v). Part of thecell suspension was pipetted on slides. Autoradiography and

determination of the number of net grains/nucleus were per-formed as described elsewhere (19), except that quantification

was done with the aid of an Artek Counter model 880 (NewBrunswick Scientific, Edison, NJ; adjusted to count mode) in

combination with a Sony CCDIRGB Video Camera (Sony,Tokyo, Japan), interfaced to a Zeiss Axioskop microscope. The

net number of grains/nucleus from a culture was obtained bycorrecting the number of nuclear grains for background grains

(by subtracting the mean number of grains in two areas equal insize to the nucleus, situated to the right and left of it). The

average number of net grains/nucleus observed in (± )-anti-

BPDE-treated cultures corrected for the average number of netgrains/nucleus observed in DMSO-treated cultures is referredto as (±)-anti-BPDE-induced UDS. Possible age-related cor-relations between ex vivo (±)-anti-BPDE-induced UDS andparameters of cytogenetic damage monitoring in vivo smoking-

induced DNA damage were statistically analyzed by means of

simple and multiple regression.

Results and Discussion

In Table 1 , means (± SD) and ranges of endogenous SCE

frequencies, MN frequencies, and (±)-anti-BPDE-inducedUDS, determined in PBLs on 2 different days from malesmokers, are shown. UDS in DMSO-treated PBLs obtained

from all individuals was very bow. No correlations were foundbetween this background incorporation and any of the cytoge-netic parameters tested or any other variables that are knownfrom these individuals. In Fig. I, results of simple regression

analysis between age of the donor and (±)-anti-BPDE-induced

UDS are shown. A significantly negative correlation was ob-served between age and (±)-anti-BPDE-induced UDS [simpleregression, r = -0.52, P = 0.019; regression equation, (±)-



35

Fig. I. Correlation between ex vito (±)-anti-BPDE-

induced UDS in PBLs obtained from male human smok-

ers and age of the donor.

30

,� 25

U

�1�I U

.,.‘ e�45 1-4

� b�10

+1 ��0

0 20 25 30 35 40 45 50 55 60

age (years)

Cancer Epidemiology, Biomarkers & Prevention 945

anti-BPDE-induced-UDS (-0.30 X age) + 33.31]. On the

basis of the regression equation, average decrease in the extentof (±)-anti-BPDE-induced DNA repair was 1. 1% per yearbetween 20 and 60 years of age. Other studies also reportnegative associations between repair, induced ex vivo in PBLswith UV, and age (5-7). Pero and Ostlund (5) and Lambert etal. (6) observed decreases in ex vivo UV-induced UDS of

approximately 0.75 and 0.43% per year, respectively, between20 and 60 years of age. Wei et a!. (4) reported a reduction inremoval of ex vivo UV-induced DNA damage of approximately

0.6% per year, within a similar range of age. However, bothpositive associations and no associations between DNA repair,determined as UV- or N-acetoxy-N-acetylaminofluorene-

induced UDS, and age have been reported as well (5, 20-22).This may be attributed to differences in either the duration ofexposure of PBLs to carcinogens or the treatment of exposedcells with radiolabeled nucleotide precursors and thus, in fact,

to differences in observed repair kinetics.(±)-anti-BPDE treatment of mammalian cells ex vivo,

including human PBLs, has been shown to result predominantly

in formation of N2-deoxyguanosine adducts [(±)-anti-BPDE-

N2-dG; Refs. 14 and 23-25]. Repair of these (±)-anti-BPDE-N2-dG adducts may proceed through transcription-coupledNER mechanisms (14, 15, 26). Recently, a mechanism fortranscription-coupled human NER was proposed (27). It isassumed that at least 17 proteins are involved in damage rec-

ognition, local DNA unwinding, dual incision around the le-sion, release of the damaged oligonucleotide and refilling of the

excision gap. In addition, (±)-anti-BPDE treatment of isolatedDNA has been shown to cause formation of base adducts like

N7-guanine and its unstable imidazole ring open product (28),which may be substrates for BER proteins in vivo. With in-

creasing age, DNA repair may decline as a consequence ofintracellular decrease in activity or levels of one or more of

these proteins involved in NER or BER.In the same individuals, no significant association was

observed between age and the rate of (±)-anti-BPDE-N2-dGadduct removal, as determined by 32P-postlabeling of DNA

obtained at different time points from isolated PBLs after a

15-mm exposure to 0.5 ,.LM (±)-anti-BPDE4 (relative meanremoval over 24 h compared to initial bevel, 7.6 ± 37%). Giventhe fact that UDS reflects the activity of all enzymes involvedin excision repair, these data suggest that enzymes that areinvolved in later steps of repair, following the initial recogni-tion and removal of (±)-anti-BPDE-induced DNA damage,

may be affected by aging.

Associations between age and initial (±)-anti-BPDE-

N2-dG adduct formation have also not been observed in thesame individuals4 [mean (±)-anti-BPDE-N2-dG adduct bevel,within 15 mm after ex vivo exposure of PBLs to 0.5 p�M

(±)-anti-BPDE, 21.2 ± 18.5 per l0� nucleotidesj. Thus, it islikely that the observed age-dependent decrease in UDS is not

related to age-dependent decreased initial (±)-anti-BPDE-DNA adduct formation. Besides, because it is conceivable that

aging results in decreased efficiency of detoxification mecha-nisms, an increase rather than a decrease in initial (±)-anti-

BPDE-DNA binding with aging would have been expected.At the individual level, total WBC count was not signif-

icantly correlated with (±)-anti-BPDE-induced UDS, exclud-

ing the possibility that intraindividual differences in effective

cellular dose of (±)-anti-BPDE was of influence on the ob-served correlation.

From this population, no information is available regard-ing T-cell subset CD4:CD8 ratios, which may be associatedwith aging (reviewed in Ref. 29). Therefore, we cannot exclude

that the observed correlation may, in case T-cebb subsets differin their sensitivity toward (±)-anti-BPDE, in fact, reflect ab-tered CD4:CD8 ratios.

In this population of smokers, interindividual variationwas observed in the extent of (±)-anti-BPDE-induced UDS,which could not be explained by smoking and alcohol andcoffee consumption. Thus, with regard to possible effects of

smoking on DNA repair, the number of cigarettes smoked perday did not correlate significantly with (±)-anti-BPDE-inducedUDS. Pack-years correlated positively with (±)-anti-BPDE-induced UDS [simple regression, r = 0.46, P = 0.042; regres-



Fig. 2. Correlation between SCE frequencies and cx1115) ( ± )-anti-BPDE-induced UDS in PBLs obtained

from male human smokers. Nu,nher.s, ages of individual

donors.

9-

8

7

6

5

1’

31

Es37 Es

33

Es � � Es3655 24-��

Es �22 35

Es�23 Es

2126 23

SCE = (-7.00 * i02� UDS) + 7.73; r=-0.45; P=0.048

0 10 15 20 25 30 35

946 Age-related DNA Damage and Repair Changes in Smokers

-

U

U

(±)-anti-BPDE-induced UDS(net grains/nucleus)

sion equation, (±)-anti-BPDE-induced UDS = (-0.29 X

pack-years) + 27.4], but when the positive correlation betweenpack-years and (±)-anti-BPDE-induced UDS was adjusted forage, this significant correlation disappeared [multiple regres-sion, r = 0.53, P 0.06; regression equation, (±)-anti-BPDE-

induced UDS = (-0.09 X pack-years) - (0.24 X age) +

32.5]. Therefore, cumulative exposure of this population tocigarette smoke does not seem to have resulted in increased

expression of those DNA repair enzymes from which the ac-

tivity can be detected by (±)-anti-BPDE-induced UDS. Fur-ther, alcohol and coffee consumption were not significantly

correlated with (±)-anti-BPDE-induced UDS [simple regres-sion, r = 0. 17, P = 0.47; regression equation, (±)-anti-BPDE-induced UDS = (0. 1 1 X number of glasses/week) + 21.7;simple regression, r 0.22, P 0.35; regression equation,( ± )-anti-BPDE-induced UDS (0.53 X cups of coffee/week)+ 20.3]. Importantly, the negative correlation between (±)-

anti-BPDE-induced UDS and age was not influenced by smok-ing habits (cigarettes/day) or alcohol and coffee consumption.

Statistical significance of the correlation between (±)-

anti-BPDE-induced UDS and age was determined by the threeoldest individuals (48, 50, and 55 years old); after these data

were omitted from statistical analysis, a negative trend was still

observed [simple regression, r = -0.30, P = 0.24; regression

equation: (±)-anti-BPDE-induced UDS = (-0.19 X age) +29.8)]. Mean (± SD) values of (±)-anti-BPDE-induced UDS asthey were determined on 2 different days from these individualswere: 14.1 ± 4.8, 14.2 ± 0.4, and 12.2 ± 3.2, respectively.

Thus, these three oldest individuals really are less proficient in

their DNA repair. It is, therefore, unlikely that the variationbetween individuals is due to variation in the used methodologyto estimate repair. Given the fact that alcohol and coffee con-sumption. smoking habits, and total WBC count were also not

associated with (±)-anti-BPDE-induced UDS and did not in-fluence the association with age, unknown factors must havecontributed to the interindividual variation observed in this

population of smokers.

Both SCE [simple regression, r = -0.45, P = 0.048;regression equation, SCE = (-7.00 X l0_2 x (±)-anti-

BPDE-induced UDS) + 7.73] and MN [simple regression, r =

-0.44, P = 0.052; regression equation, MN = (-0.14 X

(±)-anti-BPDE-induced UDS) + 8.48] negatively correlated

with the extent of(±)-anti-BPDE-induced UDS (Figs. 2 and 3).

Smoking habits (cigarettes/day) did not influence these associ-ations. Thus, the capacity of an individual’s DNA repair, as

determined ex vito in (±)-anti-BPDE-treated resting PBLs,may be indicative of the capacity of those repair mechanisms inresting PBLs in vivo, which remove those lesions that areresponsible for induction of cytogenetic damage in PHA-stim-

ulated PBLs ex vivo.

In the same population, SCE frequencies were signifi-cantly positively correlated with age [simple regression, r =

0.45, P = 0.044; regression equation, SCE = (0.041 X age)+ 4.701. MN frequencies were also positively (nonsignifi-

cantly) associated with age [simple regression, r = 0.39, P =

0.09; regression equation, MN = (0.072 X age) + 2.8 11.

Smoking habits (cigarettes/day) did not influence these as-sociations. Previous studies also report positive associations

between SCE frequencies or MN frequencies, respectively,

and age (1, 2), although no association has also been re-ported (30). Because ex vivo (±)-anti-BPDE-induced UDSwas negatively correlated with age (Fig. 1) and benzo-

(a)pyrene is a chemical constituent of cigarette smoke, it issuggested that, with increasing age, a decrease in lympho-cytic excision repair capacity may be responsible for in-creased accumulation of smoking-induced DNA lesions inthese cells. These lesions may have been responsible for theobserved age-associated increase in cytogenetic damage.Adjustment for age resulted in disappearance of the signif-

icant correlation between SCE and (±)-anti-BPDE-induced

UDS, suggesting again that the negative association between(±)-anti-BPDE-induced UDS and SCE is age dependent.

In conclusion, age-related decrease in the capacity of an

individual’s DNA repair, determined ex vivo in (±)-anti-



Fig. 3. Correlation between MN frequencies and ex

vis.o ( ± )-anti-BPDE-induced UDS in PBLs obtainedfrom male human smokers. Nu,nbers, ages of individual

donors.

10

9

8

7

6

5

4

3

2

1

0

033

035

0

� 026

48� 41� 0

50

0

22 �33 0

37 0 30

230021 23

MN = (-0.14 * UDS) + 8.48; r= -0.44; P=0.052

0 10 15 20 25 30 35

Cancer Epidemiology, Biomarkers & Prevention 947

20. Benigni, R., Calcagnile, A., Fabri, G., Giuliani, A., Leopardi, P.. and Paoletti.

A. Biological monitoring of workers in the rubber industry. II. UV-induced

©� U

a.)- a.)

U

a.)� -

U�-:

(±)-anti-BPDE-induced UDS(net grains/nucleus)

BPDE-treated unstimulated PBLs, may resemble age-relateddecrease in the capacity of repair mechanisms, acting on smok-ing-induced DNA damage in vivo. Consequently, smoking-

induced DNA damage may accumulate with increasing age andmay be revealed as an age-associated increase in SCE and MN,

after untreated PBLs are stimulated to proliferation ex vivo.

PBLs do not themselves represent target organs for carci-nogenic factors in vivo. However, in a recent study, increased

PBL chromosome aberrations were found to be associated with

increased overall risk for cancer (12). Therefore, age-dependent

decreased DNA repair and increased cytogenetic damage inPBLs may reflect similar processes that are involved in age-related development of cancer in target organs in human pop-

ulations exposed to environmental carcinogens like tobacco

smoke.

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948 Age-related DNA Damage and Repair Changes in Smokers

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1997;6:943-948. Cancer Epidemiol Biomarkers Prev R H Stierum, G J Hageman, M H van Herwijnen, et al. humans.diolepoxide-induced unscheduled DNA synthesis in smokingcytogenetic damage and ex vivo (+/-)-anti-benzo(a)pyrene Age-related negative associations between parameters of

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