32p-postlabeling assay in mice of transpiacental dna damage … · safrole treatment was estimated...

[CANCER RESEARCH 46, 3046-3054, June 19861

ABSTRACF

Transplacental exposureof fetusesto carcinogensis kno@i to inducetumors in the offspring, often with a high incidenceand short latency.While covalent adduction of DNA appears to be essential for tumorinitiation, little is known about the binding of carcinogens to the DNA offetal tissues. A sensitive32P-postlabelingmethod enabled us to study thebinding of the environmental carcinogens safrole (600 Mmol/kg p.o.), 4-aminobiphenyl (800 @imol/kg), and benzo(a)pyrene (200 ,anol/kg) to theDNA of various maternal and fetal tissues after administration of testcarcinogens to pregnant ICR mice on day 18 of gestation. The resultsshow that these carcinogens bound to the DNA of maternal and fetalliver, lung, kidney, heart, brain, intestine, skin, maternal uterus, andplacenta, with organ-specificquantitative and qualitative differences. Itwaspossiblefor the first timeto analyzeDNA adductpatternsin minuteamounts of tissue, for example those available from fetal heart. Thecovalent binding index (imol adducted nucleotides per mol of DNAnucleotides/@imolcarcinogenadministered per g body weight) 24 h aftersafrole treatment was estimated for the different organs and ranged from0.1 to 247 and 0.1 to 5.8 for maternaland fetal DNA, respectively.Covalent binding index values of 0.2 to 13 and 0.1 to 0.3 for maternaland fetal DNA, respectively, were found for 4-aminobiphenyl.Benzo(a)pyrene treatment yielded covalent binding index values ofO.6 to6.5 and 0.3 to 0.7 for maternal and fetal DNA, respectively. In bothmaternal and fetal tissues, safrole exhibited preferential binding to liverDNA. 4-Aminobiphenylbound preferentially to DNA of maternal liverand kidney but showed no preference among fetal tissues. Benzo(a)pyreneexhibited weak tissue preference in both maternal and fetal organs. Forall of thecompoundsstudied,thefetaladductlevelsweregenerallylowerthan the corresponding maternal adduct levels, especially when the levelof maternaladductionwas high. The major finding was that severalcarcinogensof diverse structure or their metabolites readily crossed theplacenta and gave rise to DNA adducts in fetal organs. The resultingDNA damage in rapidly proliferating tissues may play a critical role intransplacental carcinogenesis.

INTRODUCTION

More than 50 chemicals havebeenfound to be transplacentalcarcinogens in eight animal species(2â€”7)and in humans (8, 9),following the pioneering observation of transplacental pulmonary tumor induction by urethan (10). This suggests a possibleorigin of tumor initiation during the prenatal period (11). Fetaltissues are highly sensitive to some carcinogens (2, 3, 12â€”17).Because of increased DNA synthesis and cell proliferation ratesduring perinatal development, the threshold dose required fortumor initiation during this period may be lower than in adults,as shown by a 50-fold increase in neuro-oncogenesis by thedirect-acting ethylnitrosourea in fetal compared with adult ratbrains (18). However, an extrapolation of this observation toother classesof carcinogens requiring metabolic activation hasnot been possible (3, 7, 19â€”21)because of the differentialmaturation of drug-metabolizing enzymes during perinatal de

Received8/26/85; revised2/1 1/86; accepted2/12/86.The costsof publicationof this articlewere defrayedin part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordancewith 18 U.S.C. Section 1734solelyto indicatethis fact.

I This work was supported by USPHS grants 32157 and 13591 to K. R.Preliminarywork presented at the 76th Annual Meeting of the AmericanAssociationfor CancerResearch,Houston,TX, May, 1985(1).

2 To whom requests for reprints should be addressed.

velopment (22, 23). Therefore, a given dose of a carcinogenmay yield different amounts of reactive ultimate carcinogenand, consequently, different levels of covalent binding to DNAin an organ, depending on developmental stage. Since DNAadduction is believed to be a prerequisite for tumor initiation,it may be possible to evaluate susceptibility to tumor formationduring the perinatal period by measuring promutagenic DNAlesions.

A paucity of information exists on the covalent DNA bindingof carcinogens in fetal tissues, since most conventional fluorometric, radiometric, and spectrophotometric methods are inadequate to measure the levels of adducts found in vivo. Furthermore, someof thesemethods require mg quantities of DNAfor analysis. Therefore, to assessthe susceptibility to carcinogenesis during the perinatal period, an in vitro fetal microsomemediated mutagenesis assay (24â€”26)and a sister chromatidexchange assay using cultured embryo cells (27) were used.These experiments have provided information on fetal competence in drug metabolism. However, in vitro assaysfail to takeinto account the importance of maternal and placental factorsin transplacental carcinogenesis. Using radioactive test carcinogens, the binding of ethylnitrosourea (28), methylnitrosourea(29â€”31),BP3 (32, 33), and DMBA (34) to DNA in some fetaltissues has been shown. However, comparison between thecorresponding maternal and fetal tissues was limited to thoseorgans (liver, brain, and kidney) from which sufficient quantitiesof fetal DNA could be obtained (28, 30, 31, 34).

In the present paper, a recently developed 32P-postlabelingtest for the analysis of DNA adducts formed with non-radioactive carcinogens (35â€”39)which requires only microgramquantities of DNA was used to study DNA adduction in eightmaternal and seven fetal tissues and in placenta, elicited byexposure to the known environmental carcinogens safrole, 4-ABP, and BP (40). Safrole (41) and BP (42, 43) have beenshown to be transplacental carcinogens in mice. While this hasnot been determined for 4-ABP, another aromatic amine denyative, 2-acetylaminofluorene, is a known transplacental carcinogen (44). In view of the possible in utero exposure of humanfetusesto the three chemicals studied here and the very limitedknowledge concerning their interactions with fetal DNA, asystematic study of their binding to the DNA of maternal andfetal organs was conducted.

MATERIALS AND METHODS

Chemicals. BP, 4-ABP, and trioctanoin were obtained from SigmaChemicalCo., St. Louis, MO. Safrolewaspurchasedfrom AldrichChemicalCo., Milwaukee,WI. (Â±)BPDE I and (Â±)BPDE II were

3 The abbreviations used are: BP, benzo(a)pyrene; 4-ABP, 4-aminobiphenyl;BPDE I, (Â±)BPDE II, (Â±)DMBA, 7,12-dimethylbenz(a)anthracene;dpGp-C8-(N4-ABP),phate of N-(deoxyguanosin-8-yl)-4-aminobiphenyl; dpGp-N2-(C3-ABP), 3',S'-bisphosphateof3-(deoxyguanosin-N2-yl@4-aminobiphenyl;dpGp-N2-(C10-BPT),3',S'-bisphosphate of l0@-(deoxyguanosin-N2-yl)-7fi,8a,9a-thhydroxy-7,8,9,1O-tetrahydrobenzo(a)pyrene;PEI-cellulose,polyethylenimine-cellulose;TLC, thinlayer chromatography; D, direction or directional; C18, octadecylsilane; RAL,relative adduct labeling; CBI, covalent binding index (pmol adducts per molDNA-P/@tmolcarcinogenper g bodyweight);IF, intensificationfactor.

3046

32P-Postlabeling Assay in Mice of Transpiacental DNA Damage Induced by the

Environmental Carcinogens Safrole, 4-Aminobiphenyl, and Benzo(a)pyrene'

Lee-Jane W. Lu,2 Rose M. Disher, M. Vijayaraj Reddy, and Kurt Randerath

Department ofPharmacoIo@j@,Baylor College ofMedicine, Houston, Texas 77030

on April 22, 2020. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

Table I Intensijlcationfactorsfor safrole-, 4-ABP-, andBP-DNAadducts(RAL)

wasobtained by labelingunder intensificationconditionsat 1.7MMI-y32PJATP(4000 Ci/mmol), 400 @MDNA-P. RAL was derived by labelingunderstandard

conditions at 120 zM ATP (110 Ci/mmol), 100 @iMDNA-P(39).Carcinogen

AdductÂ° (RAL) x l0@ RAL x 10@IFbSafrole

1 9947Â±1206c 1102Â±1679.023499Â±262 348Â±6210.13

310Â±121 30.4Â±2.610.24-ABP

1 204 Â±22 95 Â±5.52.12130Â±26 7.9Â±0.816.53

25.5 Â±13.3 1.6 Â±0.215.9413.3Â±5.2 1.2Â±0.511.1BP

1 21.7Â± 1.6 3.0Â±0.77.2245.3Â±4.1 7.0Â±0.96.5318.3Â±3.0 1.0Â±0.518.343.3Â±0.6 1.5Â±0.62.257.5Â±1.5 0.4Â±0.1 18.7

32P POSTLABELING ASSAY OF FETAL DNA DAMAGE

purchased from Midwest Research Institute, Kansas City, MO. Allother materials used were as described previously (38, 45).

Animals and Treatment. Male and female ICR mice (25â€”30g) wereobtained from Harlan Co., Houston, TX. They were housed in steelcages in rooms with temperature control (20â€”22'C) and a 12-h dark!light cycle.The micewerefedFormulablaboratorychowandprovidedwith tap water ad libitum. Mating was performed once between 4:30p.m. and 8:00 a.m. Only female mice with positive vaginal plugs wereusedfor experiments. The day a positive plug wasfound wasdesignatedas day 0 of pregnancy.The gestationalperiod for ICR mice wasapproximately 19 days. Pregnant mice (2â€”4per group, â€”50 g) weretreated p.o. with safrole (600 @imol/kg),4-ABP (800 @mol/kg),or BP(200 @mol/kg)dissolved in trioctanoin (4 mI/kg) between 9:00 and11:00 a.m. on day 18 of gestation. The mice were killed by cervicaldislocation 24 h after carcinogen treatment. Fetuses (male and female)were removed by cesarean section, euthanized with CO2, and kept atâ€”80Cuntil tissue isolation.

In Vivo Modificationof DNA with BPDEI or BPDEII. Dorsalskinof non-pregnantfemaleICR micewasshavedwith electricclippersandpainted with 150 @gof(Â±)BPDE I or (Â±)BPDE II dissolved in 0.2 mlofacetone containing 10%tetrahydrofuran. Mice were killed 24 h laterby cervical dislocation, and DNA was isolated from treated whole skin.

32P-Postlabeling Analysis of DNA Adducts. DNA was isolated by aprocedureinvolvingproteinandRNA digestionandsolventextraction(39). DNA adducts were analyzed by 32P-postlabeling assay (35â€”38)with some modification (39). Labeling was done under carrier-freeintensification conditions (using a limiting amount of ATP) (39) orunder standard conditions (using excess ATP) (35, 36). The labelingmix contained 400 @M(deoxyribonucleoside 3'-monophosphate + adducted deoxyribonucleoside 3'-monophosphate), 1.7 @M[-y-32PJATP(4000 Ci/mmol) for the former and 100 @M(deoxyribonucleoside 3'-monophosphate + adducted deoxyribonucleoside 3 â€˜-monophosphate),120 MMATP (1 10 Ci/mmol) for the latter conditions. The intensification conditionswereusedhereprimarily, becausea greatersensitivity(â€”10-fold,see Table 1) was achieved by omitting non-radioactive ATPfrom the 32P-labeling mixture and performing the labeling reaction withlimiting amounts of high specific activity [â€˜y-32PJATP,as detailed elsewhere(39). The adductlevel valuesobtainedunder theseconditions((RAL)) were then divided by an IF to yield adduct levels (RAL)equivalent to those determined under standard labeling conditions (39).To obtain the IFs, a highly adducted DNA preparation from eachcarcinogen treatment was labeled under intensification as well as standard conditions (39). The IF was then defmedas (RAL)/RAL (39).Table 1 shows the IFs used for deriving the adduct levels for the 3

carcinogens studied here. The IFs for individual adducts did not varyovera l0@-foldrangeof adductlevels(39).

Mapping of32P-labeled Adducts. Because the three carcinogens studiedherehavedifferentdegreesof aromaticcharacter,whichdeterminestheir binding affinity for PEI-cellulose, the chromatographic conditionsfor both adductpurification and separationwereadjusted.Adductsformed from safrole, the least aromatic compound, were found to havethe least affinity for PEI-cellulose TLC; it was therefore necessary topurify the adducts by reversed-phase TLC (C18)(45). In the case of 4-ABP and BP adducts, purification by PEI-cellulose TLC was adequate(38). A modification of this procedure, which was found convenientand which also resulted in reduced radioactive background levels, wasused in the present work, as shown in Fig. 1. A fraction (71%) of the32P-labeled digest was applied at 2 cm from the bottom ofa 14-cm PEIcellulose sheet. Several labeled digests were applied on one sheet, 1.5â€”2 cm apart from each other. To remove normal nucleotides, developmentwasperformedovernightwith 2.4 M sodiumphosphate,pH 6.0,onto a 20-cm Whatman 1 wick attached to the top. The sheet was thencut @-10cm from the bottom, and its upper portion and the wick werediscarded.The lower portion wassoakedin 500 ml deionized water for5â€”7mm and dried with cool, then warm air. The sheet was autoradiographedfor 10â€”15mm at room temperatureusingDu Pont Cronex4 film to visualize the origin areas. The shaded areas (0.8 cm wide and1.5â€”2.5cm long; see Fig. 1) were cut out and placed in contact, layeragainstlayer,with a PEI-celluloseacceptorsheet(12x 12cm;seeFig.2). During subsequent development for adduct resolution (D3; see Ref.37), this assembly was held together between button type magnets,enablingquantitativetransferof nucleotidesfrom thedonorcut-outtothe acceptor layer. The previously described development in D2 (37)was omitted.

Because of the different adduct aromaticities, the solvent strengthsused for adduct separation in D3 and D4 were adjusted. Chromatographic conditions for safrole and BP adducts were essentially asdescribed (38, 45). For 4-ABP adduct separation, 3.0 M lithium formate:7.1 M urea, pH 3.5, was used in D3, followed by 0.6 M LiCl:0.4M Tris-HCI:6.4 M urea, pH 8.0, in D4, i.e., at a right angle to theprevious development. An additional development in 1.0 M sodiumphosphate, pH 6.8, was performed in D4 to 7 cm onto a Whatman 1wick for removalof radioactivebackgroundmaterialfrom chromatogramsof4-ABP- or BP-adducts.The 32P-labeledadductswere detectedby autoradiography ofthe chromatogram and then assayed by excisingand counting the areascontaining adduct spots for radioactivity in ascintillation counter, as described (37).

For identification of BP adducts,32P-labeleddigestsof selectedDNAs of mice treated with BP and of skin DNAs of mice treated

,â€˜#__@â€”â€”â€”â€”

I

II

II

/ /4-PAPER WICK

4ORIGINS

Fig. 1. Scheme ofadduct purification by one-dimensional PEI-cellulose TLC.32P-Labeled DNA digests were applied at least 1.5 cm apart to the origins (Aâ€”C)of a 14-cm PEI-cellulose sheet. Normal nucleotides were removed by chromatography in 2.4 Msodium phosphate, pH 6.0, overnight onto a Whatman 1 wick, asindicated(Dl). Origin areas containingpurified adducts (shadedareas) werecutout and used for subsequentadduct separation (seeFig. 2).

Safrole adductsl,2, and 3 from maternal liver DNA were identifiedas(trans-isosafrol-3'-yl)dpGp, M-(safrol-l'-yl)dpGp, and M-(trans-isosafrol-3'-yl)dpAp,respectively.4-ABPadductsI and 2 from maternalliver DNA wereidentifiedas dpGp-C8-(N4-ABP)and dpGp-N2-(C3-ABP),respectively. The identities of the remaining spots were not known, but they may be ring-openedderivatives of adduct 1. BP adduct 1 from maternal intestine DNA was identifiedas dpGp-N2-(Cl0-BPT). The remaining spots were not derived from either BPDEI or BPDE II.

b IF, intensification factor = (RAL)/RAL.C Mean Â± SD.

3047

tDI



was found to bind to all DNAs analyzed without any significantdifferences in the relative proportions of adducts, spot 1 beingthe major adduct (>70%). Among maternal and fetal tissues,safrole binding varied up to 2500-fold and 50-fold, respectively,with liver DNA being affected most strongly and brain DNAleast strongly. In several tissues, safrole bound more highly tomaternal DNA than to the corresponding fetal DNA, exhibitingthe greatest difference (42-fold) in liver but little difference inlung, brain, and intestine. Very low binding (CBI @.-0.2)to DNAof placenta and maternal uterus was also detectable. Adductswere not determined in fetal uterus.

4-ABP Adducts in Maternal and Fetal DNAs. Typical fingerprints derived from the DNA of maternal and fetal organs after4-ABP treatment are shown in Fig. 4. Maternal liver and kidney(Fig. 4, A and B) gave darker spots than did the correspondingfetal tissues (Fig. 4, C and D). Spots 1 and 2 were identifiedpreviously as dpGp-C8-(N4-ABP) and dpGp-N2-(C3-ABP), respectively (38, 47). Spots 3 and 4 might be the imidazole ringopened derivatives of adduct 1 (data not shown).

As shown in Table 3, 4-ABP bound to DNA from all maternaland fetal tissues analyzed, with spot 1 being the major adduct(80â€”90%of total) at the time point examined. In maternaltissues, 4-ABP bound most strongly to DNA ofliver and kidneyand least strongly to brain DNA (range of binding, â€˜-.â€˜70-fold).On the other hand, little preferencewas observedfor individualfetal tissues (range of binding, <4-fold). 4-ABP bound morestrongly to some maternal tissues than to the correspondingfetal tissues, especially in kidney (70-fold) and liver (48-fold),while maternal and fetal DNA-binding values were similar forbrain and lung. Placental DNA showed low levels of 4-ABPadducts.

(B) BP Adducts in Maternal and Fetal DNAs. 32P fingerprints of

DNA adducts from maternal and fetal organs ofmice 24 h afteradministration of BP are shown in Fig. 5. In contrast to theother two compounds investigated, adduct patterns variedgreatly among the maternal organs studied, as illustrated byexamples shown in Fig. 5, A to D. Little variation was noted,on the other hand, among the fetal DNAs (Fig. 5, E to H). Spot1 was identified as dpGp-N2-(C10-BPT) (see below). Identitiesof the remaining spots were unknown.

Quantitative data ofthe covalent binding of BP to DNA fromvarious maternal and fetal tissues are listed in Table 4. Amongmaternal tissues, total BP-DNA binding varied up to 10-fold,while the major adduct (No. 1) varied <2.4-fold. The numberand relative proportions ofthe BP adducts obtained were tissuedependent, with the most complex pattern noted for intestinalDNA (Fig. SC). Spot 1 was the major spot, except for intestinalDNA, where spot 2 predominated. Among the various fetaltissues,BP adduct levelsvaried <1.8-fold in terms ofboth totaland adduct 1 binding. Spot 1 was the major adduct in all fetalDNAs and, in most cases, it was the only spot detected. Totalfetal adduct levels were 1.6â€”15-foldlower than were the comesponding maternal levels. However, the amount of adduct 1 inall fetal tissues investigated was -â€˜-3-foldlessas compared withthe corresponding maternal DNA, except for a 1.6-fold difference observed between maternal and fetal brain DNA. BPbound to placental DNA at a slightly higher level than it did tothe other fetal DNAs.

In an attempt to identify the spots obtained from BP-DNA(Fig. 5), 32P-labeleddigests of intestinal DNA of BP-treatedpregnant mice were co-chromatographed with 32P-labeledBPDNA adduct markers, which were prepared from the skin ofmice treated topically for 24 h with BPDE I or BPDE II. Theresults ofco-chromatography are shown in Fig. 6. Fig. 6, A and


I

D3t

ORIGIN

@,@@-DONOR CUT@OUT

â€¢2@4â€” 2 â€”â€˜

Fig. 2. Scheme for adduct separation by two-dimensional PEI-cellulose TLC.

A cut-outcontainingthe origin area(Fig. I) wasattachedto an acceptorPEIcellulose sheet (12 x 12 cm) with the aid of 2 button-type magnets for contacttransfer of adducts. Adduct separation was performed by development in Liformate/urea, pH 3.5 (D3), followed by LiC1/Tris-HCI/urea, pH 8.0 (D4).

topically with (Â±)BPDE I or (Â±)BPDE II were mixed and cochromatographed. Chromatographic conditions were as describedabove for BP adduct analysis, except that the solvent for D4 development was a mixture of 2-propanol and 4 N NH.OH (1:1, vol/vol).

Estimation of Extent of DNA Adduction. The count rate (cpm) ofeachdetectableadductspot wasevaluatedas described(37, 38, 45).RAL was calculated according to the formula (39):

RAL â€”cpm in adducted nucleotides mol of adducts (A)â€” cpm in total nucleotides x IF mol of DNA-P

TheCBI wascalculatedby:

CBI â€”@imoladducted nucleotides/mol DNA-P PAL x 10'â€” Dose (pmol carcinogen/g body weight) Dose

RESULTS

The three environmental carcinogens, safrole, 4-ABP, andBP, were found by 32P-postlabeling assay to bind covalently toDNA of maternal and fetal liver, lung, kidney, heart, brain,intestine, and skin, as well as to maternal uterine and placentalDNA. Fetal adduct levels were generally lower than were maternal adduct levels. The assayenabled us to detect and quantitate DNA adducts in minute amounts of tissue available suchas fetal heart, with six fetal hearts (wet weight, <10 mg) yielding-@-70@tgDNA. This was more than sufficient for replicate adductanalysis, which required a total of <15 @tgDNA.

$afrole Adducts in Maternal and Fetal DNAs. Fig. 3 showstypical fingerprints of safrole-DNA adducts, derived from maternal and fetal organs. Fingerprints of safrole adducts werequalitatively similar in all examples shown (Fig. 3, Aâ€”F), aswell as in the other maternal and fetal organs investigated (notshown). Spots 1, 2, and 3 were identified previously as N2-(trans-isosafrol-3'-yl)dpGp, N2-(safrol-1 â€˜-yl)dpGp,and N6-frrans-isosafrol-3'-yl)dpAp (45, 46). Spot 4 was a small amountof a 3'-dephosphorylated derivative of spot 1 (data not shown);thus, for calculations, count rates of this spot were added tothose of spot 1. The extent of dephosphorylation varied withbatches of C18reversed-phase TLC plates. Fig. 3, A and D,represents autoradiograms exposed for -@-45mm, while autoradiography for the other samples was for 32 h at â€”80Â°C.Inspection of the autoradiograms suggeststhat adduct levels inmaternal and fetal liver exceededthose in the other tissues.

Quantitative data of adduct levels and CBI values for variousmaternal and fetal organs analyzed are given in Table 2. Safrole

3048



TissuesOrigin4PAL

xl0@'RAL,@,JRAL,CBrSpot

ISpot 2Spot3TotalLiverM

F1102 Â±167'

26.4 Â±3.6348Â±61.5

7.5 Â±1.130.4Â±2.6

1.0 Â±0.241480Â±154

35.0 Â±3.142.32475.8LungM

F2.1Â±0.23

1.9 Â±0.320.79Â±0.13

0.41 Â±0.250.13Â±0.12

0.14 Â±0.153.0Â±0.44

2.5 Â±0.581.20.500.42KidneyM

F4.1Â±0.34

1.3Â±0.181.4Â±0.16 0.37Â±0.140.21Â±0.11

0.12Â±0.065.7Â±0.59 1.8Â±0.353.20.950.30HeartM

F2.6Â±0.40

1.2Â±0.371.1Â±0.52

0.37Â±0.100.07Â±0.01

0.18Â±0.073.7Â±0.84

1.7Â±0.462.20.620.28BrainM

F0.49Â±0.05

0.56Â±0.190.07Â±0.04

0.09Â±0.080.04Â±0.01

0.03Â±0.030.60Â±0.08

0.69Â±0.270.80.100.12IntestineM

F1.5Â±0.21

1.1 Â±0.420.36Â±0.13

0.32Â±0.110.08Â±0.02

0.04Â±0.012.0Â±0.35

1.5Â±0.521.30.330.25SkinM

F5.1Â±0.64

0.71 Â±0.141.6Â±0.53

0.16Â±0.080.26Â±0.08

0.04Â±0.026.9Â±1.1

0.92Â±0.227.51.20.15UterusM'1.0

Â±0.070.34 Â±0.050.05 Â±0.01.4 Â±0.020.23Placenta1.2

Â±0.250.17 Â±0.060.02 Â±0.021.4 Â±0.010.23


CA

. S..,

Fig. 3. Fingerprints of â€œP-labeledsafroleDNA adducts from variousmaternal and fetalmouse organs treated on day 18 of gestationwith safrole (600 gimol/kgp.o.) for 24 h. Adducts were purified by C13reversed-phase TLCand separatedby 1.8 M Li formate/4.25 Murea, pH 3.5 (D3) and 0.36 M LiCI/0.22 MTris-HCI/3.8 M urea, pH 8.0 (D4) (45). Spots1.2, and 3 representN2-(trans-isosafrol-3'-yl)-dpGp, N2-(safrol-l â€˜-yl)dpGp,and N'-(transisosafrol-3'-yl)dpAp.Spot 4 is a dephosphorylated derivative of spot 1. Weak spots arecircled. Autoradiography at â€”80Â°Cwas for 45mm (A and D) or 32 h (B, C, E, and F).

Bâ€¢.4

3-ft

i@2 VIe.

..2

MATERNAL LIVER

Q4

SI

D

3*Q

s-I

FETAL LIVER FETAL KIDNEY FETAL BRAiN

Table 2 Safrole-DNAadduct levelsin variousmaternal andfetoJ tissuesEachpregnantmousewasgivensafrole(600 @mol/kgp.o.)onday18ofgestation,andDNA wasisOlated24h later.

aM,maternaltissue;F,fetaltissue.aFordefinitionseeTable1.CCBI = @smoladducts per mol DNA.P/pmol carcinogen per g mouse.d Mean Â± SD.

V Adduct level in fetal uterus was not determined.

B, shows that BPDE I and BPDE II treatment each resulted in

one major adduct (>98%), spots a and c, respectively. Thesolvent used for D4 of the chromatograms shown in Fig. 6 wasa 1:1 (vol/vol) mixture of 2-propanol and 4 N NI{.OH, replacing the previously published LiCI/Tris/urea solvent (38) becauseof better resolution of the major BPDE I adduct (spot a)from the major BPDE II adduct (spot c) (Fig. 6C). Fig. 6Dshows a 32P-labeled adduct pattern of BP-DNA from mouseintestine in this system. Since development in D3 for the mapsshown in Fig. 6D and Fig. SC was performed in the same

solvent, spots shown in the two figures were cross-referencedvia their mobilities in D3 and by scintillation counting (datanot shown). It can be seen (Fig. 6D) that spot I migrated fasterin the 2-propanol/NHOH solvent than did spots 2 and 3. Fig.6, E and F, exemplifies co-chromatography of BP adductsobtained from intestinal DNA of pregnant mice (Fig. 6D) withBPDE I (Fig. 6A) or BPDE II adducts (Fig. 6B) derived frommouse skin. Adduct a (Fig. 6A) was found to co-chromatographwith adduct I (Fig. 6D), as shown in Fig. 6E. Adduct c (Fig.6B) did not co-chromatograph with any of the spots seen on

3049

MATERNAL KIDNEY MATERNAL BRAIN

E â€¢. FQ..4



TissuesOrigin'RAL

x107bRALMJRALFCBICSpot

1Spot 2Spot 3Spot4TotalLiverM

F95.4Â±55'

1.9Â±0.647.9Â±0.84

0.16Â±0.041.6Â±0.18

0.07Â±0.031.2Â±0.45

NCC106Â±3.1

2.2Â±0.6648.213.30.28LungM

F3.5Â±0.18 1.3Â±0.210.19Â±0.030.11 Â±0.010.10Â±0.050.03Â±0.010.13Â±0.09 NC3.8Â±0.31 1.4Â±0.202.70.480.18KidneyM

F72.9Â±5.6

0.85 Â±0.143.9Â±0.94

0.13 Â±0.051.5Â±0.34

0.08 Â±0.020.85Â±0.23

NC78.9Â±6.4

1.1Â±0.1671.79.90.14HeartM

F7.7Â±0.83 0.46Â±0.140.46Â±0.020.06Â±0.010.16Â±0.010.06Â±0.010.14Â±0.02 NC8.4Â±0.81 0.57Â±0.1215.01.10.07BrainM

F1.3Â±0.20 0.72 Â±0.140.07Â±0.030.03 Â±0.010.04Â±0.020.05 Â±0.01NC NC1.5Â±0.20 0.80 Â±0.141.90.190.10IntestineM

F12.1Â±0.51

0.85Â±0.110.78Â±0.05

0.14Â±0.050.25Â±0.04

NC0.27Â±0.06

NC13.4Â±0.51

0.99Â±0.1413.51.70.12SkinM

F12.7Â±1.2

1.1 Â±0.220.52Â±0.110.08 Â±0.010.30Â±0.150.01 Â±0.00.34Â±0.180.02 Â±0.013.9Â±1.4

1.2 Â±0.2311.61.70.15UterusM'5.4Â±0.170.22Â±0.050.17Â±0.050.07Â±0.015.8Â±0.170.73Placenta1.7

Â±0.420.15 Â±0.060.03 Â±0.01NC1.9 Â±0.360.24C

NC, not countable.


Similar observations were made for adduct I from all othermaternal and fetal organs of mice treated with BP (data notshown). Chromatographic evidence thus indicated that adduct1 was dpGp-N2-(C10-BPT) (48, 49). This was confirmed by cochromatography in two additional solvent systems (data notshown).

In order to facilitate a comparison of DNA binding of thethree carcinogensstudiedhere, the CBI valuesobtainedwerearranged as shown in Fig. 7. A wider range of CBI values wasfound for safrole or 4-ABP than for BP, but in general, in bothmaternal and fetal DNA, liver was at or close to the top, andbrain was at or close to the bottom of the CBI scale. The CBIvalues were usually higher for maternal DNA than for thecorresponding fetal DNA, but after safrole treatment, a considerable number of fetal tissuesexhibited higher CBI values thandid some maternal tissues (Fig. 7).

DISCUSSION

The 32P-postlabeling assay has been used in this study toshow that the carcinogens safrole, 4-ABP, and BP crossed theplacenta readily, giving rise to measurable DNA adducts inevery fetal tissue examined. DNA binding was also detected inevery maternal tissue analyzed. These observations may helpexplain the reported transplacental carcinogenicity of safrole(41) and BP (42, 43) in mice. The transplacental carcinogenicityof 4-ABP was not known; however, unpublished experimentsshowed that 4-ABP bound to fetal tissue DNA to a similarextent as did a related transplacental hepatocarcinogenic aromatic amine derivative, 2-acetylaminofluorene (44). The strongpreferenceof safrolefor bindingto fetal liver overkidneyDNA,when administered as a single dose in late gestation, was not inline with the reported transplacental renal carcinogenicity afterchronic administration throughout gestation (41). Safrole, however,is a hepatocarcinogenwhengiven to newbornmice (50,5 1). BP, administered as multiple doses during mid-gestation,preferentially induced lung adenomas and, if promoted postnatally with croton oil, elicited skin papillomas in the offspring(42, 43). We observed that BP bound to all fetal tissues tosimilar extents. Taken together, our data suggestedthat initial

A B

4

MATERNAL LIVER MATERNAL KIDNEY

C 2@ @D 20

. â€œ.3 . â€˜3I I I

@frâ€¢

FETAL LIVER FETAL KIDNEYFig. 4. Fingerprintsof32P-labeled4-ABP-DNA adductsfrom various maternal

and fetal mouseorgans treated on day 18 ofgestation with 4-ABP(800 gimol/kgp.o.) for 24 h. Adducts were purified by PEI-cellulose TLC(Fig. I) and separatedby 3.0 M Li formate/7.l M urea, pH 3.5 (D3) and by 0.6 M LiCl/0.4 M Tris.HCI/6.4 Murea, pH 8.0 (D4). Spots I and 2 representdpGp-C8-(N'-ABP)and dpGpN2-(C3-ABP), respectively. Spots 3 and 4 appeared to be the ring-opened derivativesofadduct 1.Weakspotsarecircled.Autoradiographyat â€”SOTwasfor 3 h(A and B) or 17 h (Cand D). Spots in upperright-handcornerofCand D werenotderivedfromadducts.

Fig. 6D (Fig. 6F). These results suggestedthat adduct 1 was aderivative of BPDE I. As shown in Fig. 6, G and H, the majorBP-DNA adduct (spot 1) from maternal liver and fetal lung alsoco-chromatographed with the BPDE I-derived adduct (spot a).

Table 3 4-ABP-DNA adduct levels (ML x 1O@)in various maternal andfetal tissuesEach pregnant mousewas given4-ABP(800 @mol/kgp.o.) on day 18 ofgestation, and DNA was isolated24 h later. For Footnotes a to e, see Table 2.

3050



TissuesOrigin'RALxI0@'RAL,@JRAL,CBrSpotISpot 2Spot 3Spot 4Spot5TotalLiverM

F2.6Â±0.27d@

0.84 Â±0.401.2Â±0.26

NC0.28Â±0.09

NC0.68Â±0.25

NCNC' NC4.9Â±0.74

0.84 Â±0.405.82.50.42LungM

F2.8Â±0.35

0.98 Â±0.250.28Â±0.08

NC0.35Â±0.08

0.12 Â±0.010.72Â±0.08

NCNC NC4.2Â±0.39

1.1 Â±0.253.82.10.55KidneyM

F1.6Â±0.20

0.68 Â±0.020.23Â±0.15

NC0.10Â±0.04

NC0.41Â±0.22

NCNC NC2.4Â±0.33

0.68 Â±0.023.51.20.34HeartM

F2.8Â±0.30

0.97Â±0.16NC NC0.22Â±0.05

NC0.18Â±0.13

NCNC NC3.3Â±0.24

0.97Â±0.163.41.70.49BrainM

F1.2Â±0.210.76 Â±0.08NC NCNC NCNC NCNC NC1.2Â±0.210.76 Â±0.081.60.600.38IntestineM

F3.0Â±0.680.73Â±0.157.0Â±0.910.10Â±0.071.0Â±0.49 NC1.5Â±0.56 NC0.37Â±0.11 NC13.0Â±1.9

0.84Â±0.1115.56.50.42SkinM

F1.8Â±0.29

0.63 Â±0.15NC NC0.05Â±0.02

NCNC NCNC NC1.9Â±0.30

0.63 Â±0.153.00.950.32UterusM'1.5Â±0.56NC0.13Â±0.120.23Â±0.13NC1.9Â±0.430.95Placenta1.2Â±0.120.10Â±0.010.08Â±0.01NCNC1.3Â±0.110.65e

NC, not countable.

32pPOSTLABELINGASSAYOF FETALDNA DAMAGE

I@.

f

FETAL INTESTINE FETAL HEART

A B C

, -.3Fig. 5. Fingerprints of 32P-labeled BP

DNA adducts from variousmaternal and fetalmouse organs treated on day 18 of gestationwith BP(200 @mol/kgp.o.)for 24h. Adductswere purified by PEI.cellulose TLC (Fig. 1)and separated by 3.6 M Li formate/8.5 M urea,pH 3.5 (D3) and 0.8 M LiC1/0.5 M Tris-HCI/8.5 M urea, pH 8.0 (D4). Spot 1 was identifiedas dpGp-N2-(Cl0-BPT). Identities of the remaining spots were unknown. The unmarkedspots were not derived from adducts. Autoradiographyat â€”80Cwas for 24 h (Aâ€”E)or 2days (Fâ€”H).

MATERNAL INTESTINE MATERNAL HEARTMATERNAL LUNG MATERNAL LIVER

E 1'-

@..

FETAL LUNG FETAL LIVER

03

H

S.-'

e@.

Table 4 BP-DNA adduct levels in various maternalandfetoJ tissuesEach pregnant mouse was given BP (200 @mol/kgp.o.) on day 18 ofgestation, and DNA was isolated 24 h later. For Footnotes a to e, see Table 2.

A B C

Fig. 6. Co-chromatography of 32P-labeledBP-DNA adducts from maternal and fetal tissues (Fig. 5) with 32P-labeledBPDE I- orBPDE lI-DNA adducts from mouse skin.SourcesofDNA wereas indicated.32P-labeleddigests from various preparations were mixedand applied to PEI-celluloseTLC for purification (Fig. 1). Adduct separation was as inFig. 5, except that the D4 solvent was replacedwith a 1:1 mixture of 2-propanol and 4 NNH4OH. Spots 1-4 designate DNA adductsderived from mice treated with BP. Spots aand b are DNA adducts derived from skin ofmice treated topically with (Â±)BPDE I, andspots c and d are from (Â±)BPDE Il-treatedmice. Spots a-d are deoxyguanosine adducts(data not shown). Autoradiography at â€”80Â°Cwas for 60 h (A, B, D-F, and H), 15 h (C), or40h(G).

â€¢0

H

I44-MATERNAL LI ER

8+0 BR-DNA

3051

G@@

MATERNAL INTESTINEBRâ€”DNA

A+FETAL LUNCBR-DNA

BPDEI-DNABPDE Ilâ€”DNA4+B@gbâ€¢2F@;C@1

jOb




values have beenreported for 4-ABP in adult mouse tissues forcomparison with the present data. BP bound most strongly tomaternal intestinal DNA, in line with data reported for the rat(57). CBI values of BP in maternal tissues determined herewere comparable with literature data (58).

As to the possible relationship between the level offetal DNAbinding and transplacental carcinogenic potency, few data areavailable in the literature for comparison. From published data,we estimated CBI values of @-i0for ethylnitrosourea (28) andDMBA (34) each, and @@-300for methylnitrosourea (31), ofwhich >90% was due to the non-mutagenic 7-methylguanine

@.-IflTEarINE adduct, in the major target organ, fetal rat brain. The fact that

ethylnitrosourea is a potent transplacental neuro-carcinogen inspite of its low CBI value suggestedthat a low CBI value forfetal DNAs may be highly significant in terms of the postnatalexpression of prenatal DNA damage. The CBI values for fetalmouse DNAs observed here after safrole, 4-ABP, and BPtreatment implied that these compounds may be weaker transplacental carcinogens overall than are ethylnitrosourea, methylnitrosourea, and DMBA, but this comparison may be compromised because of animal species and treatment scheduledifferences.

The three carcinogens studied here require different modesof metabolic activation (5 1, 59). While BP is activated viaoxidation by mixed-function oxidases, the activation of safroleand 4-ABP involves additional conjugation reactions (Si, 60).The differential enzyme requirements for biotransformation ofthe three carcinogens were reflected by the complex tissue- anddevelopmental age-specific adduct profiles shown here for eachof the carcinogens studied (Fig. 7).

The exceptionally strong preference of safrole for fetal liverDNA suggested that safrole readily crossed the placenta andthat fetal liver actively participated in its bioactivation, in linewith the reported high sulfotransferase activity for i â€˜-hydroxysafrole right after parturition in mice (6i). 4-ABP exhibited astrong preference for binding to maternal liver and kidneyDNA, but unlike safrole, the maternal pattern of omganotropismwas not apparent among fetal tissues, indicating immaturity ofactivation enzymes or efficient DNA repair in the fetal tissues.The uniform fingerprints of safrole and 4-ABP adducts suggesteda single major ultimate carcinogen for thesecompoundsin fetal and maternal tissues, in accord with published observations for adult tissues (51). The strong organotropism ofsafrole and 4-ABP binding may be related to quantitative vanations of activation and detoxification in different tissues. Because the two carcinogens showed a strong preference formaternal tissues, a first-pass effect may have reduced theamounts ofthe carcinogens and/or their active metabolites thatwere available for transport across the placenta to the fetuses.This, coupled with the immaturity of fetal activation enzymes(22, 23), may havebeenresponsiblefor the relatively low CBIvalues for safrole and 4-ABP in fetal tissues.

The great variation of BP-DNA adduct patterns among thevarious maternal tissues and the demonstration that only oneBP-DNA adduct was from the bay-region diol-epoxide pathwaywere consistent with the existence of more than one pathwayof metabolic activation of BP (62). Since adduct 1 co-chromatographed with the major BPDE I-DNA adduct from mouseskin and also with the 32P-labeledderivative of the reactionproduct of BPDE I with dGp,4 spot 1 was the BPDE I derivative, dpGp-N2-(CiO-BPT) (48, 49). Since the remaining BPDNA adducts(2â€”5)did not co-chromatographwith any markeradducts prepared in vitro or in vivo from BPDE I or BPDE II,their identities were unknown. However, the reduced chromat

o2_

i@L

$00.

â€˜0-I

@--LIVER

.---. liver

rSKIN.--KIDNEYâ€˜@-HEART

@.--lung,â€˜@[email protected]::â€”[email protected]

-â€˜@ UTERUS

â€˜[email protected]

.. brain

â€˜BRAIN

xw0z

(9z0zcc

zU-J

>00

..--. LIVER

. .--- KIDNEY

,-SKIN@---INTESTINE

.. --HEART

-UTERUS.-LUNG

r--liver@ ,placenta

@â€œ.lung@@.BRAIN

..-skin:@kidney@,:-intestine

.- brain

-heart

,LIVER-:.-LuNGL.â€”HIART

-KIDNEY@-SKIN@UTERUS@placenta

@.BRAIN

;t\@lung@heart

[email protected]@@-intest inc@-brainkidney

kâ€”skin

SAFROLE 4-ABP BPFig. 7. Levels of DNA binding as expressed by the covalent binding index

(CR1) of safrole, 4-ABP, and BP in various maternal and fetal tissues CBI isdefined as @zmoladducts per mol DNA-P/@molcarcinogen per g body weight.Upper case letters refer to maternal DNA, and lower case letters refer to fetalDNA.

DNA bindingper se may not be a completely accurate predictorof the target organ for tumor formation in the offspring. Thismay be related to differences in the strains of mice used, indosing schedules,or in DNA repair among various fetal tissues.As shown especially for ethylnitrosourea in rats (52â€”54;forreview, seeRef. 55), persistenceof the initially formed adductsafter transplacental carcinogen dosing may determine organspecific tumor formation in the offspring.

In terms of maternal DNA binding, safrole and 4-ABPshowed the strongest preference for liver DNA, while BPshowed little preference for any tissue. This is in line withsafrole and 4-ABP being liver carcinogens and BP being asystemic carcinogen in adult mice (40, 51). The maternal andfetal results taken together suggested that DNA binding datamay serve to indicate carcinogenic potential of a compound(56) at different developmental stages but fail to predict accurately the organ site(s) of tumor formation in either mother orprogeny. It appears that additional variables not studied hereneed to be taken into account, such as individual or total adductpersistence and promotional influences.

In order to facilitate comparisons of hepatocarcinogenic potency among various chemicals, Lutz (56) defmed a CBI, witha CBI value between 10 and 100, suggesting a weak hepatocarcinogen and a CBI value in the hundreds, suggestinga moderatehepatocarcinogen. According to this hypothesis, safrole wouldbe a moderate, 4-ABP a weak or borderline, and BP a nonhepatocarcinogen in the pregnant ICR mice. The relationshipof CBI values to organ-specific carcinogenic potency has notbeen established for tissues other than liver. The CBI value forliver DNA of the pregnant ICR mice was 3â€”8-foldhigher thanthat reported for non-pregnant CD-i (45) or ICR mice,4whichsuggested that safrole may be a more potent hepatocarcinogenin pregnant ICR mice than in non-pregnant mice. No CBI

41.-i. W. Lu and K. Randerath, unpublished observation.

3052




5. ivankovic, S. Chemical and viral agents in prenatal experimental carcinogenesis. Biol. Res. Preg., 3: 99â€”102,1982.

6. Kleihues,P. Developmentalcarcinogenicity.In.@K. Snell,(ad.),DevelopmentalToxicology,pp.213â€”246.NewYork: Praeger,1982.

7. Rice, J. M. Exposure to chemical carcinogens during pregnancy. consequences for mother and conceptus. In.@R. A. Pattillo and R. 0. Hussa (ads.),Human Trophoblast Neoplasms, pp. 13â€”49.New York: Plenum PublishingCorp., 1984.

8. Greenwald,P., Barlow,J. J., Nasca,P. C., and Burnett,W. S. Vaginalcancerafter maternal treatment with synthetic estrogens. N. EngI. J. Med., 285:390â€”392,1971.

9. Herbst,A. L., and @lly,R. E. Adenocarcinomaofthe vaginain adolescence:a report of 7 cases including 6 clear cell carcinoma (so-called mesonephro.mas).Cancer (Phila.), 25: 745â€”757,1970.

10. Larsen, C. D. Pulmonary-tumor induction by transplacental exposure tourethane. J. Nail. Cancer Inst., 8: 63â€”69,1947.

11. Miller, R. W. Prenatal origins of cancer in man: epidemiologicalevidence.IARC Sd. PubL,4: 175â€”180,1973.

12. Druckrey,H. Chemicalstructure and action in transplacentalcarcinogenesisand teratogenesis. IARC Sd. Publ, 4: 45â€”58,1973.

13. Kleihues, P., Mende, C., and Reucher, W. Tumors of the peripheral andcentralnervoussysteminducedin BD-ratsbyprenatalapplicationof methylmethanesulfonate.Eur.J. Cancer,8:641â€”645,1972.

14. Herbst, A. L., Cole, P., Colton, T., Robboy,S. J., and Scully, R. E. Ageincidence and risk of diethylstilbestrol-related clear cell adenocarcinoma ofthe vagina and cervix. Am. J. Obstet. GynecoL 128: 43â€”50,1977.

15. Napalkov,N. P., and Anisimov,V. N. Transplacentaleffectof diethyistilbestrol in femalerats. Cancer Left, 6: 107â€”114,1979.

16. Nomura, T. Carcinogenicity ofthe food additive furylfuramide in foetal andyoungmice.Nature (Lond.), 258: 610â€”611, 1975.

17. Rustia, M., and Shubik, P. Transplacental effects ofdiethylstilbestrol on thegenital tract ofhamster offspring.Cancer Lets., 1: 139â€”146,1976.

18. Ivankovic, S., and Druckrey, H. Transplacentare Erzeugung Maligner TumorandesNervensystems.L Athyl-nitrosoharnstoffan BD-IX Ratten.Z.Krebsforsch.,71:320-360, 1968.

19. Clayson, D. B. Carcinogenesis in the developing organism: could protocolsfor testing be improved?Biol.Res. Preg, 2: 150â€”158,1981.

20. Rice, J. M. An overviewof transplacentalchemicalcarcinogenesis.Teratology,8:113â€”126,1973.

21. Rice, J. M. Perinatal period and pregnancy intervals ofhigh risk for chemicalcarcinogens. Environ. Health Perspect., 29: 23â€”27,1979.

22. Dutson, G. J., and Leakey, J. E. A. Drug metabolizing enzymes. Prog DrugRes.,25:191â€”275,1981.

23. Lucier, G. W., Lui, E. M. K., and Lamartiniere, C. A. Metabolic activation!deactivationreactions during perinatal developmenLEnviron. Health Perspect., 29: 7â€”16,1979.

24. Blake, D. A., Martz, F., Gery-Martz, A., Gordon, G. B., and Mellits, E. D.Fetal tissuesfrom variousstrains of inducedmicemetabolizebenzo(a)pyreneto mutagenicmetabolites.Teratology,20: 377â€”382,1979.

25. Juchau, M. R, DiGiovanni, J., Namkung, M. J., and Jones, A. H. Acomparison of the capacity offetal and adult liver, lung, and brain to convertpolycyclicaromatic hydrocarbonsto mutagenicand cytotoxicmetabolitesinmiceand rats. Toxicol.Appl. PharmacoL,49: 171â€”178,1979.

26. Sehgal, C. B., and Hutton, J. J. Homogenates of pregnant rat and fetaltissuesmetabolizecarcinogensto mutagensdetectedby Salmonellatyphimurium TA98 and TA100. Mutat. Res, 46: 325-344, 1977.

27. Galloway, S. M., Perry, P. E., Meneses, J., Nebert, D. W., and Pedersen, R.A. Cultured mouse embryos metabolize benzo(a)pyrene during early gestation:geneticdifferencesdetectablebysisterchromatidexchange.Proc.NatL.@cad.Sci.USA, 77:3524â€”3528,1980.

28. .Ã³th, R., and Rajewsky,M. F. Ethytationof nucleicacids by ethylnitrosoureft-l-â€•Cin the fetal and adult rat. Cancer Res.,32: 1501â€”1505,1972.

29. Wunderlich, V., and Tetzlaff, I. Alkylierung der Kern DNA verschiedenerOrgane der Ratte durch Nitrosomethylharnstoff in sivo. Arch. Geschwulstforsch.,35:251â€”258,1970.

30. Magee,P. N. Mechanismsof transplacentalcarcinogenesisby nitroso cornpounds. IARC Sci. Pubi, 4: 143â€”148,1973.

31. Likhachev, A. J., Alexandrov, V. A., Anisimov, V. N., Bespalov,V. G.,Korsakov,M. V., Ovsyannikov,A. I., Popovic,I. G., Napalkov,N. P., andTomatis,L Persistenceof methylatedpurinesin the DNA of variousratfetal and maternal tissues and carcinogenesis in the offspring following asingle transplacental dose of N-rnethyl-N-nitrosourea.Int. J. Cancer, 31:779â€”784,1983.

32. Wunderlich, V., and Tetzlaff@I. Applikation von (3H@benzo(a)pyrenan grayide MIUSÃund Ratten: Inkorporation der RadioaktivitIt in subzellulÃ£reFraktionen verschiedener Gewebe. Arch. Geschwulstforsch., 42: 95â€”106,1973.

33. Shum, S., Jensen, N. M., and Nebert, D. W. The murine Ah locus:in uterotoxicity and teratogenesis associated with genetic differences inbenzo(a)pyrenemetabolism.Teratology,20: 365-376, 1979.

34. Doerjer, G., Diessner, H., BUcheler, J., and Kleihues, P. Reaction of 7,12-dimethylbenz(a)anthracenewith DNA of fetal and maternal rat tissues inpivo.Int J. Cancer,22: 288â€”291,1978.

35. Randerath, K@,Reddy, M. V., and Gupta, R. C. 32P-Postlabeling test forDNA damage.Proc. NatI. Aced.Sd. USA, 78:6126â€”6129,1981.

36. Reddy, M. V., Gupta, R. C, and Randerath, K. â€œP-Baseanalysis of DNA.Anal. Biochem., 117: 271â€”279,1981.

37. Gupta, R. C., Reddy, M. V., and Randerath, K 32P-Postlabeling analysis of

ographic mobilities of adducts 2 and 3 in the 2-propanol/NH4OH solvent would appear to be consistent with phenolicderivatives (62). The surprisingly high level of adduct 2 inmaternal intestinal DNA may have been due to the formationof an active metabolite by intestinal flora.

The formation of BPDE I from BP is dependent on cytochrome P-450 linked monooxygenase (for review, seeRef. 59).While this enzyme has been reported to be deficient in extrahepatic tissues of rodents (22, 23, 63), in our experiments,adduct I levels showed little difference among the maternal andamong the fetal DNAs. The similarity in BP adduct I levels indifferent tissues was also observed in â€¢non-pregnant mice andmale rabbits (58). These data suggestedsimilar concentrationsof BPDE I in different maternal and fetal tissues, respectively.These results may be explained by possible tissue-specific differences in DNA repair, deficiency of detoxifying enzymes,and/or BPDE I synthesis in maternal liver with transport toother tissues.

In vitro assays of mixed-function oxidases (22, 63) and ofmicrosome-mediated mutagenesis (24â€”26)with rodent adultand fetal tissues have shown the latter to be 1 to 2 orders ofmagnitude lessactive than the former. The fact that our bindingdata for the BPDE I-derived adduct 1 did not reflect thisdifference was not surprising, since the in vivo assayevaluatedthe net result of metabolic activation, detoxification, and DNArepair, and, in addition, considered the roles played by placentaand maternal tissues in carcinogen activation and delivery.

In conclusion, we have presented comprehensive covalentDNA-binding data for three structurally diverse chemicals, i.e.,safrole, 4-ABP, and BP, in maternal and fetal organs. Theresults showed that (a) fetal DNA was damaged quite extensively in utero by these carcinogens; (b) DNA damage wasdetected in every tissue examined; (c) maternal DNA adductlevels were generally higher than were fetal adduct levels; (d)fetal tissues probably participated in the biotransformation ofthe chemicals to reactive metabolites; (e) the resulting DNAadducts were tissue- and developmental age-specific, indicatingthat DNA-binding data from adults cannot be extrapolated toprenatal DNA; and ( f) carcinogens requiring conjugation formetabolic activation (safrole and 4-ABP) showed stronger organotropism of DNA binding than a carcinogen not requiringconjugation (BP). Binding to fetal DNA appeared to be theresult ofa combination ofmaternal, fetal, and possibly placentalfactors. It may be hoped that the DNA-binding data presentedhere will provide a basis for the future quantitative assessmentof relationships between prenatal DNA adduction and carcinogenesis in the offspring.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Erika Randerathfor stimulatingdiscussions.We aregratefulto Mark E. Schurdakfor critical readingof the manuscript.

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3053



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1986;46:3046-3054. Cancer Res Lee-Jane W. Lu, Rose M. Disher, M. Rijayaraj Reddy, et al.

)pyrenea4-Aminobiphenyl, and Benzo(Induced by the Environmental Carcinogens Safrole,

P-Postlabeling Assay in Mice of Transplacental DNA Damage32

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