Persistenceof Benzo(a)pyreneMetabolite:DNAAdductsin Lungand Liverof MiceMahmooda S. Kulkarni1 and Marshall W. Anderson
Laboratory of Pharmacology, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina 27709
ABSTRACT
The persistence of benzo(a)pyrene (BP) metabolite:DNA ad-
ducts has been studied in lung and liver of A/HeJ and C57BL/6J mice after a dose of BP (6 mg/mouse) which induces pulmonary adenomas in A/HeJ mice but not in C57BL/6J mice. BP isnot a hepatic carcinogen in either strain. Following p.o. administration of [3H]BP, animals were killed at times ranging from 10
hr to 28 days, and BP metabolite: DNA adducts were analyzedby high-pressure liquid chromatography. The major adduct identified in each tissue was the (+)-7/3-8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:deoxyguanosine adduct. A7/3,8a-dihydroxy-9|8,10/3,epoxy-7,8,9,10-tetrahydrobenzo(a)py-renerdeoxyguanosine adduct, a (-)-7/3,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene:deoxyguanosine ad
duct, and an unidentified adduct were also observed. The disappearance of (+)-7;8,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tet-rahydro-BP adduct in A/HeJ mice followed first-order kineticsover the time period examined, with a half-life of 18 and 9 days
in lung and liver, respectively. The decay of this adduct in C57BL/6J mice was biphasic in both tissues. Our data on cell turnoversuggest that there is active removal of adducts in liver, but thatnormal DMA turnover can account for the partial or possibly totalobserved disappearance of adducts in lung. These results suggest that the tissue specificity for BP-induced neoplasia in A/HeJ
mice may be related to the relative persistence of adducts andhigh cell turnover rates in lung. In contrast, the results onformation and persistence of adducts and cell turnover do notprovide an explanation for the strain difference in susceptibilityto BP-induced pulmonary adenomas. It was also shown that the
rates of removal of BP metaboliteiDNA adducts in A/HeJ miceare not significantly different at a 500-fold lower BP dose.
INTRODUCTION
Mutation and malignant transformation of cells by chemicalsmay be a consequence of DNA synthesis on parent-strand
templates containing unexcised chemically induced lesions. Theability of a cell to repair the damaged DNA by an error-free
pathway prior to cell replication could constitute a critical protective mechanism against mutagenesis and carcinogenesis (5,11,14). Yang et al. (36, 37) demonstrated a linear relationship innormal human fibroblasts between the number of BPDEI2:DNA
adducts present at the time of DNA replication and BPDEI-
induced mutation frequency. If the cells were allowed to remainin confluence until the BPDEI adducts had been removed byexcision repair, then no mutations were detected. In contrast,the length of time that repair-deficient xeroderma pigmentosum
cells remained in confluence had no effect on the frequency ofBPDEI-induced mutations. In vivo evidence suggests that thedeficient DNA repair processes found in individuals with xeroderma pigmentosum (13, 30) and ataxia telangiectasia (26) predispose them to neoplasia. Also, some investigators have suggested that the persistence of specific carcinogen:DNA adductsin the target tissue correlates with the susceptibility of the tissueto neoplasia. For example, the persistence of O6-ethylguanine
adduct appears to correlate with the susceptibility of rat brain toneoplasia induced by ethylnitrosourea (20). Eastman and Bres-nick (15) claim that 3-MC metabolite: DNA adducts are morepersistent in lungs of mice strains susceptible to PAH-induced
pulmonary neoplasia than in resistant strains, and Abbott andCrew (1) argue that the persistence of DNA adducts formed frommetabolites of 15,16-dihydro-11-methylcyclopenta(a)phenan-threne-17-one, together with the relatively high rate of cell division, may be related to the tissue-specific carcinogenesis of this
polycyclic ketone in TO mice.PAHs such as BP are a major class of ubiquitous environmen
tal carcinogens. Recent studies have characterized the in vivoformation of PAH metabolite:DNA adducts in a variety of tissues(1,4,6,9,15,17,22,29). However, there have been few studieson the persistence of these adducts (1,15, 29). The persistenceof BP metabolite:DNA adducts in vivo in mouse skin after topicalapplication of BP has been examined (29), but no correlationwas observed between persistence of adducts and susceptibilityof various strains of mice to PAH-induced neoplasia, a result that
pearance of adducts in lung and liver of A/HeJ mice was alsoexamined at a much lower dose of BP in order to assess theeffect of initial BP metabolite: DNA adduct levels on persistenceof the adducts. To determine the contribution of normal DNAturnover to the observed in vivo rates of disappearance of DNAadducts, we also measured the rates of DNA turnover in thevarious tissues.
' To whom requests for reprints should be addressed, at Laboratory of Phar
macology, NIEHS/NIH, P. O. Box 12233, Research Triangle Park, N. C. 27709.'The abbreviations used are: BPDEI, (±)-7/S,8a-dihydroxy-9a,10o-epoxy-
Received April 7,1983; accepted September 28,1983.
MATERIALS AND METHODS
Chemicals. [G-3H]BP (specific activity, 56 Ci/mmol) and unlabeled BP
were obtained from Amersham Corp. (Arlington Heights, III.) and SigmaChemical Co. (St. Louis, Mo.), respectively. The fluorescent dye H33258was purchased from Calbiochem-Behring Corp. (La Jolla, Calif.). Hydrox-ylapatite (DNA grade; Bio-Gel HTP) was obtained from Bio-Rad Labora
tories (Richmond, Calif.). Calf thymus DNA, alkaline phosphatase type III
(EC 3.1.3.1), and phosphodiesterase I (EC 3.1.4.1) were purchased fromSigma. DNase I was purchased from Worthington Biochemical Corp.(Freehold, N. J.) [3H]Thymidine (specific activity, 60 to 80 Ci/mmol) was
obtained from New England Nuclear. All other chemicals were reagentgrade or better.
Animals. Seven- to 9-week-old female A/HeJ and C57BL/6J mice
(The Jackson Laboratory Bar Harbor, Maine) were used in all experiments. The mice were randomly selected by age before allocation totreatment groups. They were fed NIH-31 rat and mouse ration (Zeigler
Brothers, Inc., Gardner, Pa.) and given water ad libitum for the durationof each study.
Treatment of Animals for Adduct Disappearance Studies. The BPdosing regimen is based on a previous study of Wattenberg (33) for theinduction of pulmonary adenomas in A/HeJ mice. A/HeJ and C57BL/6Jmice received a total of 6 mg of BP administered as 2 equal p.o. doses(2x3 mg) 2 hr apart. BP dose consisted of 3 mg of unlabeted BP and 1mCi of [3H]BP dissolved in 0.25 ml com oil. Animals were killed by
cervical dislocation at times ranging from 10 hr to 28 days followingtreatment with BP (15 animals for each time point). The lungs and liverswere removed and frozen in liquid nitrogen. In another experiment withA/He J mice, a much lower BP dose (0.012 mg/mouse) was used. Animalsreceived a total of 2.6 mCi of |3H|benzo(a)pyrene, administered as 2
equal p.o. doses 2 hr apart.Isolation and Analysis of BP Metabolites Bound to DNA. DNA was
isolated from each pooled tissue sample by the hydroxylapatite procedure described previously (2). Samples of the purified DNA were assessed for DNA content by using the fluorescent dye (H33258) adductmethod of Cesarone ef al. (12). Calf thymus DNA was used as a referencestandard. UV absórbanos measurements were also made using therelationship 1 mg DNA = 20 Ajeo units.
DNA was digested enzymatically to deoxyribonucleosides and theirBP metabolite adducts according to the method of Baird and Brooks (7).These adducts were analyzed by a modified version of the HPLC andliquid scintillation counting procedure discussed previously by Andersonet al (4). The HPLC system (Waters Associates, Milford, Mass.) consistedof a dry-packed precolumn containing the same material as that used ina Waters 10 /IM C-18 Radial Pak analytical column. The precolumn was
freshly packed and washed with methanol before each sample run. Theelution program was carried out at a flow rate of 0.8 ml/min. The sampleand internal standards (acetophenone, butyrophenone, and BP-9,10-diol)
were loaded on to the precolumn and washed with 100% water for 10min followed by methanol:water containing 0.1% triethylammonium acetate, pH 7, (40:60) for 9 min. Fifty drops were collected per vial. Thesesamples were called the water fractions. The precolumn, containingretained adducts, was then connected to the analytical column, and thegradient fraction eluted with a linear methanol:water:triethyl-ammonium
acetate gradient (40 to 70% in 40 min). Fifty drops/vial were still collecteduntil after the first internal standard peak (acetophenone) was seen. Thedrop number was then changed to 15 drops/vial throughout the remainder of the run.
Reference standards of BP metabolite:deoxyribonucleoside adductswere prepared by incubation of [G-3H]BP with 3-MC-induced liver micro-
somes in the presence of DNA as described by Pelkonen ef al. (27).Other standards were also prepared by reacting [G-3H](±)-7/3,8a-dihy-droxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene and BPDEII
with DNA as described by Yamasaki ef al. (35). Chromatograms of thereference standards are given by Anderson ef a/. (4).
Rate of DNA Turnover in Lung and Liver. Mice received [3H]thymidine
(15 itCi/mouse i.p.) on 2 consecutive days (1). After 3 days, one groupof animals was killed (zero time point), and the remaining animals receivedunlabeled BP (6 mg/mouse) or vehicle. Mice were killed at times rangingfrom 1 to 21 days following BP administration (5 animals/time point).DNA was isolated from lung and liver and quantitated as describedabove. Aliquots of isolated DNA samples were measured for radioactivity.Labeled DNA (dpm/mg DNA) at various time points was compared tothe amount of DNA labeled at zero time point. Relative percentage ofloss of labeled DNA was used as a measure of DNA turnover.
98
RESULTS
Disappearance of BP Metabolism:DNA Adducts in MouseLung and Liver. The rate of disappearance of BP metabolite-
DNA adducts in lung liver of A/HeJ and C57BL/6J mice wasstudied after a single p.o. dose of [3H]BP (6 mg/mouse). This
dose, administered twice, 2 weeks (3), or 6 weeks apart (33),induced 21 (3) or 17 (33) pulmonary adenomas/mouse in A/HeJmice. Animals were sacrificed at times ranging from 10 hr to 28days following p.o. administration of BP. The specific activitiesof BP metabolite:deoxyribonucleoside adducts were determinedby HPLC analysis. A typical HPLC analysis is shown in Chart 1.Four peaks, labeled I, II, III, and IV, were observed in each tissueand in each mouse strain. Peaks II and III have been identifiedpreviously as (-)-BPDEI:dGuo and (+)-BPDEI:dGuo, respec
tively. Peak IV was identified as a BPDEIhdGuo adduct. Peak Icould be either a benzo(a)pyrene-phenol-oxide:DNA adduct or aBPDEI :deoxycytidine adduct. In addition to Peaks I to IV, un-
characterized radioactivity eluted in the WF and early in themethanohwater gradient (4,15).
The time-dependent decrease in (+)-BPDEI:dGuo adduct lev
els (Chart 1, Peak III) in lung and liver of A/HeJ and C57BL/6Jmice after a p.o. dose of BP is shown in Chart 2. In A/HeJ mice,this BPDEI adduct decayed exponentially in both lung and liver,the half-life being 18 and 9 days, respectively (Chart 2). In
contrast, the BPDEI adduct levels in C57BL/6J mice did not
GF
1200 r
1000
800
I 60°
400
200
0
LUNG
9, lOdiol
20 40 60
FRACTION NUMBER
GF
80
500
400
300
200
100
O
LIVER
20 40 60 80
FRACTION NUMBER
Chart 1. HPLC of BP metabolite:deoxyribonucleoside adducts in lung and Tverof mice. Mice were killed 48 hr after a p.o. dose of [3H]BP (6.0 mg/mouse). DNA
isolated from these tissues was enzymatically digested, and the deoxyribonucleosides were chromatographed on HPLC as described in •Materialsand Methods."
Peaks are discussed in the text. Arrows, positions of the internal standards. WF,water fraction; GF, gradient fraction; BPN, butyrophenone; AP, acetophenone.
Chart 2. Disappearance over time of BPDEI:DNA adduci from lung and liver ofA/HeJ and C57BL/6J mice. Mice were sacrificed at times ranging from 10 hr to 28days following p.o. administration of [^JBP (6 mg/mouse). DMA isolated from lungor liver was enzymatically digested, and the deoxyribonucleosides were chromat-ographed on HPLC. The specific activity (pmol/mg DMA) of the BPDEhDNA adduciwas calculated from the area under Peak III in a chromatogram similar to thosepresented in Chart 1. Points, average of 2 HPLC determinations.
decay in a monophasic manner (Chart 2). The same generalkinetics were observed in a repeat experiment in which adducilevels were only determined at 1, 7, 14, and 28 days followingBP dose (data not shown). In the repeat experiment, BPDEIadduct levels decayed exponentially in lung and liver of A/HeJmice with a half-life of 17 and 13 days, respectively, and the
decay of this adduct in C57BL/6J mice was not monophasic. Inlung of A/HeJ mice, the disappearance of BPDEII (Peak IV) andPeak I adduct levels paralleled the decrease in BPDEI adductlevels. The half-lives for removal of BPDEII and Peak I adducts
in lung of A/HeJ mice were 14 and 17 days, respectively. Theseadducts could not be quantitated in liver over the entire timespan. (We did not attempt to determine the specific activity of apeak unless the counts in the peak were at least 100 dpm abovebackground.)
The disappearance of BP metabolite: DNA adducts in lung andliver of A/HeJ mice was also examined at a much lower dose ofBP (0.012 mg/mouse) to determine if the rates of disappearancefor the adducts depended on the initial levels of the adduct. Aswith the higher dose of BP, the BPDEI adduct levels decayedexponentially (Chart 3). The half-lives for loss of the adduct were
17 and 13 days in lung and liver, respectively. Although the initialBPDEI adduct levels in lung and liver at the higher BP dose wereapproximately 1700- and 2700-fold larger, respectively, than at
the lower dose, the removal rates of the adduct were similar atthe 2 dose.
DNA Turnover Rates in Lung and Liver of Mice. The rate ofDNA turnover in lung and liver of BP-treated and control micewas measured by prelabeling the DNA with [3H]thymidine. No
difference in DNA turnover rates was observed in BP- andvehicle-treated mice. After BP treatment, there was an initialrapid loss of [3H]thymidine from lung in both A/HeJ and C57BL/
6J mice (Chart 4). Approximately 7 days after BP treatment, theamount of pH jthymidine in lung appeared to either approach a
0.005
1
~ 0.001
yS 0.002
0.001a
Q0003
LUNG
T,,,'17days
LIVER
T,/2=13doys
12 7 14DAYSAFTER BPADMINISTRATION
28
ChartS. Disappearance of BPDEhDNA adduct from lung and liver of A/HeJmice. Mice were sacrificed at times ranging from 10 hr to 28 days following p.o.administration of pH]BP (0.012 mg/mouse). DNA isolated from lung and liver wasenzymatically digested, and the deoxyribonucleosides were chromatographed onHPLC. The specific activity (pmol/mg DNA) of the BPDEI.-DNA adduct was calcu
lated from the area under Peak III in a chromatogram similar to those presented inChart 1. Points, average of 2 HPLC determinations.
¿'00
S 80
< 60
Z 40
ÓÃ*
< 20O
Z
1 100ÕÕ80
| 60
2 401-
20 •
A/HeJ
C57BL/6J
2 6 10 14 18 22TIME AFTER BP TREATMENT (DAYS)
Chart 4. Loss of |3H|thymidine-labe!ed DNA from lung and liver of A/HeJ and
C57BL/6J mice over 21 days following treatment with BP (6 mg/mouse). DNA wasprelabeled with (3H|thymidine as described in "Materials and Methods.' After
administration of unlabeled BP (6 mg/mouse), animals were sacrificed at timesranging from 1 to 21 days. DNA was isolated from lung and liver, and specificactivity (dpm/mg DNA) was determined. Ordinate, amount of DNA labeling ispercentage of label at time of BP administration.
constant level, being approximately 20 to 30% of the level attime of BP treatment, or to decrease at a much slower rate(Chart 4). [3H]Thymidine levels in liver of C57BL/6J mice also
decreased after BP dose and then approached a constant levelof 60% of the initial level. In contrast, [3H]thymidine levels in liver
of A/HeJ mice did not decrease significantly for the first 7 daysafter BP dose. The levels then decreased to 50% of initial level.Each time course for [3H]thymidine disappearance was done in
duplicate. The values at each time point are averages from the2 separate experiments. The shapes of the time curves for[3H]thymidine disapperance in the 2 experiments were very
similar.
DISCUSSION
We examined the formation and disappearance of BP metab-
olite:DNA adducts in lung and liver of A/HeJ and C57BL/6J mice.We also compared the rate of cell turnover in these tissues andstrains of mice. Although adducts disappeared at detectablerates in both the tissues, it is not possible at present to concludethat the removal in lung is due to DNA repair processes. Thenormal rate of DNA turnover could account for the partial orpossibly total observed disappearance of adducts in lung of bothstrains. The relative rate of loss of [3H]thymidine-labeled DNA in
lung of both strains was greater than the rate of decay of theadducts as shown in Charts 2 and 4. Since there is very littleDNA turnover in liver of A/HeJ mice for the first 7 days followingBP dose, the removal of adducts in this tissue may be due toDNA repair. Although there is some DNA turnover for the first 4days in liver of C57BL/6J mice, the initial loss of adducts is morerapid as well as sustained for the entire period of study. Abbottand Crew (1) examined the removal of DNA adducts formedfrom metabolites of 15,16-dihydro-11-methyl-cyclopenta(a)-phenanthren-17-one and DNA turnover rates in lung, skin, and
liver of TO mice. Their data also suggest that there is a activeremoval of adducts in liver, whereas normal DNA turnover couldaccount for the removal of adducts in lung and skin. If adductsdo persist in some specific cell types in lung (skin) but areremoved by enzymatic repair in the liver before cell division, thenthis, in part, may account for the susceptibility of lung (skin) andresistance of the liver to PAH-induced neoplasia in A/HeJ and
TO mice. Several investigators (21, 23, 24) have induced liverhepatomas and enzyme-altered foci in rats when the PAH is
administered after partial hepatectomy. Thus, PAH can be carcinogenic in the liver under altered conditions of cell replication.
On the contrary, our data on formation and persistence of BPmetabolite-DNA adducts and on cell turnover rates offer noexplanation for the strain difference in susceptibility to BP-
induced pulmonary adenomas. Although the disappearancecurves for adducts in lung are distinctly different in A/HeJ andC57BL/6J mice, one being a first-order decay and the other a
biphasic decay, the levels of adducts are similar initially as wellas at 28 days following the BP dose. Conclusions similar to ourswere reached by several other workers. Phillips ef a/. (29) studiedthe formation and disappearance of DMBA metabolite:DNA adducts in skin of several mice strains. Their data could not explainthe strain difference in susceptibility to DMBA-induced neoplasiain skin of mice. Similar conclusions were reached with BP and3-MC, although adduct levels were examined at only 2 time
points (29). Pelkonen ef al. (27) studied the disappearance of BPadducts in skin and s.c. tissue of C3H and C57BL/6J mice. Therate of disappearance of the adducts did not differentiate between the C57BL/6J resistance and the C3H susceptibility toBP-induced s.c. fibrosarcomas. In contrast, Eastman and Bres-nick (15) reported that the persistence of 3-MC metabolite:DNAadducts in mouse lung correlated with susceptibility of the various mice strains to 3-MC-induced pulmonary adenomas. The
reasons for the discrepancy between the results of Eastman andBresnick (15) and the other studies on correlation between
persistence of PAH metabolite:DNA adducts and strain susceptibility to PAH-induced neoplasia is unclear.
It should be emphasized that the specific activities of the PAHmetabolite-DNA adducts reported in this and the above-men
tioned studies are calculated on the basis of the total DNA in theorgan. It is possible that the amounts of adducts formed as wellas their repair rates in different cell types of the target organmay vary considerably. Several investigations have demonstrated cell specificity in the in vivo formation and repair ofcarcinogen:DNA adducts in liver for 1,2-dimethylhydrazine and2-acetylaminofluorene (8, 32, 34). Correlations were observed
between the persistence of adducts and cell specificity for livercarcinogenesis (8, 32). Examination of the formation and persistence of PAH metabolite:DNA adducts in individual cell typesof the target tissue might allow differentiation of tissues withrespect to susceptibility and resistance to PAH-induced neopla
sia.The persistence of BP metabolite: DNA adducts in lung and
liver of A/HeJ mice was examined at both a carcinogenic doselevel and at a 500-fold lower dose. No significant diffference in
the rates of disappearance was observed, even though therewas a 700-fold difference in the initial levels of the adducts.
Consideration of the dose dependency of adduct formation andpersistence is important in the low-dose extrapolation of carci
nogenic data (5, 15, 28), and we know of no previous study onthe dose dependency of the persistence of PAH metabolite: DNAadducts in vivo.
The excisability and persistence of BPDE:DNA adducts havebeen studied in several cell culture systems (10,11,16,18,19,31, 36, 37). In these studies, DNA turnover was accounted forby keeping the cells in confluence (36, 37), or by prelabeling thecellular DNA prior to BPDE treatment (11, 16, 18, 19, 31) and,thus, the removal of adducts can probably be equated withexcision repair. Feldman et al. (18) found the excisability ofBPDE:DNA adducts in a human lung tumor cell line to be poor,and 50% persisted for several generations. They speculated thatthe persistent adducts could have resulted from the loss ofexcision repair capacity during prolonged incubation or from thelocation of the adducts on a portion of the DNA that cannot berepaired by the excision pathway. Eastman et al. (16) madesimilar observations in hamster trachea! epithelial cells.
In conclusion, although the persistence of carcinogen metabolite-DNA adducts in tissues with high DNA turnover is conducive
for the initiation of neoplasia, the persistence of the adducts perse is apparently not sufficient for the induction of neoplasia.Elucidation of the protective role of excision repair in PAH-induced neoplasia requires further examination in target andnontarget tissues.
ACKNOWLEDGMENTS
The authors thank Catherine White, Julie Angerman-Stewart, Karen Galloway,and Gillian Comyn for their excellent technical assistance. They are also grateful toDebbie Gamer who typed the manuscript.
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