[CANCER RESEARCH 37. 1490-1496, May 1977]

gens transform cells to a malignant state, which involveschanges in gene expression (8, 41). They cause immediatechanges in gene transcription (4, 26, 27), including activation of viral genes (40), and induce chromosomal abemmations(20, 21). Consequently, adinect action ofthese PAH onthe genetic material, as occurs with steroid hormones (18,37), is suspected. If this is the case, studies on their nuclearinteraction would be the 1st step in explaining the alterationof gene expression by these compounds. One logical objective would be to identify specific interactions of carcinogensin the nuclei of intact transformable cells.

Previous studies demonstrated that about 2 to 5% of the

whole cell [3H]MC is found in the isolated detergent-treatednuclei during a 1- to 24-hr exposur€.of the cells to theradioactive compound (29). Approximately 90% of this nuclear-bound radioactivity is associated with chrornatin

(deoxynibonucleoproteins) isolated from these nuclei. Themajority of the radioactivity in the nucleus after a 4-hr exposure of the cells to [3H]MC is localized in 1 subnucleanfraction (Fraction I) isolated on sucrose gradients (29).

Compared to other fractions, this fraction contains the majonity of the rapidly labeled nRNA and the highest levels ofderepressed DNA (e.g., for DNA-dependent RNA synthesisin an in vitro system) and acidic (nonhistone) proteins (42).

Fraction I represents 5 to 15% of the total nuclear DNA, withthe other fractions representing 85 to 95% of the total cellDNA. These ranges of DNA represent variations betweenexperiments. At low doses of [3H]MC, Fraction I shows a100-fold greater binding than the other fractions.Interestingly,the bindingto FractionI is saturable,

whereas that to the other fractions is not saturable. Inaddition, Fraction I also displays a similar affinity forDB(a,h)A, another carcinogenic PAH, but shows little binding to DB(a,c)A, a weakly carcinogenic PAH (29). In short,there appears to be a specific, high-affinity binding of[3H]MC and/on its metabolites to a particular nuclearsubfraction (Fraction I) after 4 hr of exposure to AKR mouseembryo cells(29).

Since carcinogenic PAH have been reported to requiremetabolic alterations before they are capable of causingmalignant transformation (17, 32, 43) and since previousstudies on transformation of cells in culture involved exposure periods longer than 4 hr, we decided to reassess the

nuclear binding to Fraction I with longer exposure periodsso that the metabolism of PAH becomes more evident. Thepresent study compares the nuclear binding of [3H]MC inthe same cell culture systems as described previously (29,42) with longer periods of exposure. Data on the binding

SUMMARY

A high-affinity localization of [3H]methylcholanthreneand/or its metabolites to a specific nuclear fraction (FractionI)between4 and 72 hrofexposureisdescribed.Othercarcinogenic hydrocarbons, such as benzo(a)pynene anddibenz(a,h)anthracene , demonstrate a similar marked localization in Fraction I after 24 hr of incubation. The weak

carcinogen dibenz(a,c)anthracene, as well as steroid hormones, show little localization in this fraction. Two types ofbinding are measured: an organic solvent-extractable (noncovalent) binding and a nonextractable (covalent) binding.Maximal levels of the combined extractable and nonextractable binding per mass DNA are found at 24 hr of exposure,whileat48 and 72 hrofexposurethebindingisreduced.The highest level of the nonextractable binding pen massDNA is also observed at the 24-hr exposure period. However, as the period of exposure increases, the proportion ofthe total nuclear-bound radioactivity representing the nonextractable type increases. Analysis by high-pressure liq

uid chromatography of the extractable radioactivity fromthe fractions indicates that, the longer the period of exposure, the greater the extent of metabolism of 3@methylchol

anthrene. When 7,8-benzoflavone (a flavanoid hydroxylaseinhibitor) is included in the incubations, practically all metabolic alterations of the parent compound are prevented. Inaddition, the time-dependent increase in nonextractableradioactivity from all nuclear subfractions is prevented. Ametabolic-dependent “covalent'â€b̃inding of carcinogenicpolycyclic aromatic hydrocarbons to the various nuclearsubfnactions of chromatin is suggested. This covalent binding is markedly localized in a specific fraction of the chromatin containing rapidly labeled nascent RNA.

INTRODUCTION

One probable mechanism of action of carcinogenic PAH4involves an alteration of gene expression. These cancino

†T̃his investigation was supported by Grants CA-14920 and CA-16816

from the National Cancer Institute, Department of Health, Education andWelfare, and by the Mayo Foundation.@

2 To whom requests for reprints should be addressed.

3 Recipient of Contract CB-53886, under which most standard polycyclic

aromatic hydrocarbons were supplied.4 The abbreviations used are: PAH, polycyclic aromatic hydrocarbons;

MC, 3-methylcholanthrene; DB(a,h)A, dibenz(a,h)anthracene; DB(a,c)A, dibenz(a,c)anthracene; HPLC, high-pressure liquid chromatography; BP,benzo(a)pyrene; BF, 7,8-benzoflavone; 11-OH, 11-hydroxy-3-methylcholanthrene; cis-1 1.12-diol, 3-methylcholanthrene cis-1 1,12-dihydrodlol; 1112-epoxide, 3-methylcholanthrene 11.12-dihydroepoxide.

Received October 13, 1976; accepted February 14, 1977.

1490 CANCER RESEARCH VOL. 37

Effects of Metabolism on the Binding of Polycyclic

Hydrocarbons to Nuclear Subfractions of CulturedAKR Mouse Embryo Cells1

Thomas C. Spelsberg,2 Thomas H. Zytkovicz, and Harold L. Moses3

Departments of Molecular Medicine (T. C. S. , T. H. Z. Jand Pathology and Anatomy(H. L. M.J, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55901

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Nuclear Binding of MC Metabotites

Assay of Nonextractable Radioactivity. Fractions of thesucrose gradients were pooled to obtain Fraction I (Gradient Fractions 4 through 11), Fraction II (Gradient Fractions 12 through 19), and Fraction III (gradient pellet) (seeChart 1). The organic solvent-extractable radioactivity wasassessed by 2 methods. In Method I, aliquots (2 ml on more)of each of the fractions were shaken in conical glass centnifuge tubes with 3 volumes of benzene at 25°for 20 mm . Thetubes were centrifuged 2 mm at 1000 x g. The benzene layer

(upper layer) was removed , and the extraction was repeatedtwice. The pooled benzene extract was frozen and lyophilized. This extract either was used for assay of molecularspecies by HPLC, as described below, or was solubilized in10 ml of the PPO-POPOP toluene-based scintillation fluor,after which the radioactivity was measured in a BeckmanLS-100 scintillation spectrometer with a counting efficiencyof 38%. In Method II, 100- to 300-pi aliquots of each of thefractions were precipitated with 1.0 ml of cold 10% tnichloroacetic acid. The insoluble material was collected on aMillipore filter (2.4-cm diameter with 0.4-pm pore size) andwashed 2 times with 2 ml of cold 5% tnichloroacetic acid.The filters were dried under a heat lamp and placed in 5 mlof the toluene-based PPO-POPOP fluon. After 30 mm, withintermittant agitation, the vials were counted. The filterswere removed, and the vials were recounted. The countsrecorded in absence of the filter (extracted in the toluenebased fluon) represent the extractable radioactivity. Thecounts still bound to the filter (not extracted in the fluon)represent the bound or nonextractable radioactivity. Theradioactivity, extracted by either benzene (Method I) ontoluene (Method II), represents the “extractable―portion, whileresidual radioactivity represents the “nonextractable' ‘pontion. The latter activity is assumed to represent the covalentbound material, although actual evidence for this type of

binding is not presently available.HPLC. The metabolites of [3HJMCwere extracted from the

nuclear fractions with 3 volumes of benzene by Method I asdescribed above for assay of extractable radioactivity. Standands of MC metabolites were also solubilized in benzeneand treated similarly to the fraction extracts. The benzeneextracts were frozen and lyophilized. The residual materialwas solubilized in 100% acetonitrile, filtered through Millipore Fluoropore filters (1-pm pore size) (Millipore Corp.,Bedford, Mass.), and applied to a Waters Model 202 highpressure liquid chromatograph (Waters Association, Milfond, Mass.) using a reverse phase system. This systemconsisted of one 30- x 0.6-cm pBondapak C18column with aprogrammed linear gradient over a i-hr period beginning

with 30% acetonitnile/70% water (v/v) and ending with 65%

acetonitnile/35% water (v/v). Dual Model 6000 pumps withan automatic programmer (Waters Association) were usedto generate the gradients. All runs were performed at 25°at2 mI/mm for a gradient period of 60 mm [giving a change inacetonitnile concentration of 0.58% (v/v) per mini. For monitoning of the unlabeled standard metabolites of MC, an UVmonitor with a i00-@l cell was used at a wavelength settingof 2540 A. The spectrophotometer signal was recorded on aHewlett-Packard 3380A recording integrator, which registenedretentiontimesoftheelutingUV-absorbingcomponents, as well as calculated the relative abundance of eachof these UV-absorbing components (assuming equivalent

affinity and specificity with respect to the nuclear subfractions and carcinogenic versus noncarcinogenic compounds are presented. Two types of binding are quantitated: an extractable type, representing “noncovalent―binding, and a nonextractable type, representing “covalent―binding. Also shown is the requirement for metabolism of the PAH for nuclear covalent binding. The extractable metabolites are analyzed by HPLC with the use of areversephasesystem.

MATERIALS AND METHODS

Cell Culture. Descriptions of the transformable cell line(AKR-2B) and the methods of culture have been given previously (14, 29, 32, 33, 40, 42). Cells were grown in 490-sq cmplastic roller bottles (Corning) in McCoy's 5a medium supplemented with 10% fetal calf serum, penicillin G (100 units/ml), and streptomycin (100 @g/ml).From 3 to 5 bottles wereharvested by trypsinization and collected in cold completemedium for each experimental point. For the 72-hr expeniments, 15 to 25 bottles of cells were used.

Treatment with Hydrocarbons. The cells are treated withthe various PAH and processed for subsequent nuclei isolation as described previously (29). [G-3H]MC (2.2 Ci/mmole)and [G-3H]BP (20 Ci/mmole) were purchased from Amensham/Seanle, Arlington Heights, ill. [G-3H]DB(a,c)A (685mCi/mmole) and [G-3H]DB(a,h)A (2.42 Ci/mmole) were punchased from New England Nuclear, Boston, Mass. Unlabeled BP, DB(a,h)A, and MC were purchased from ICNPharmaceuticals Inc., Plainview, N. Y. DB(a,c)A and BF (a-naphthoflavone) were purchased from Nutritional Biochemicals Corp., Cleveland, Ohio, and Aldrich Chemical Co.,Inc. , Milwaukee, Wis. , respectively. The standard metabolites of MC, consisting of li-OH, cis-ii,i2-diol, 11,12-dione, 3-methyl-7-phenylacenaphthene , and 11,12-epoxidewere prepared by The Midwest Research Institute and distnibuted by the Illinois Research Institute, Chicago, III. , forthe National Cancer Institute. Additional standards of cis11,12-diol and the 11,12-epoxide were also kindly donatedby Dr. Peter Sims (Chester Beatty Institute of Cancer Research, London, England). The radioactive compoundswere tested for purity (and purified if required) with thehigh-pressure liquid chromatography, as described below.The [3H]BP was adjusted with unlabeled BP to give a specific activity of 670 Ci/mole. [3H]DB(a,c)A and [3H]DB(a,h)Awere adjusted with unlabeled material to give 233 mCi/mmole. These samples were then lyophilized and stored at—20°until needed. Lyophilized material was dissolved indimethyl sulfoxide immediately prior to use, and 35 .d wereadded to 35 ml of culture medium per roller bottle to give afinal concentration of 1 @Ci/mlon 0.4 j.tg/ml for the various[3HJPAH. The dimethyl sulfoxide concentrations in treatment and control bottles were equal. Cells were consistently treated when in a near confluent, rapidly proliferatingstate.

Isolation of Nuclei and Nuclear Fractionation. The nucleiwere isolated and fractionated, and the fractions werechemically analyzed as described previously (29, 42). Themethod is outlined in the legend of Chart 1. DNA wasquantitated by the diphenylamine method (7).

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16

8

00 24

Relative distribution of radioactivity among the nuclearfractionsPAH

added tocells%

of totalradioactivity―Hrof exposureFraction I Fraction II FractionIll[3H]MC4

24487264

26 1063 26 ii54 25 2140 2634[3H]BP2453

35i2[3H]DB(a,h)A246720i3[3HIDB(a,c)A246619 i5

T. C. Spetsberg et at.

:@H@MCextinction coefficients). Radioactive samples (or any sampies to be purified) were collected on a Gilson microfractioncollector in 0.6-mI fractions (on 18 sec/fraction). For monitoning of radioactive components, each of the 0.6-mI fractions was solubilized in iO ml of toluene-based PPO-POPOPscintillation fluor containing iO% Biosolve (Beckman). The

relative abundance of each of the eluting radioactive components was achieved by dividing the radioactivity in eachpeak by the total radioactivity of all peaks eluted during thechromatographic run. For purification of unlabeled stan

dands, the fractions containing the UV-absorbing peakaround the desired retention volume were pooled and lyophilized.

RESULTS

Binding to the Nuclear Subfractions during Varying Exposure Periods. Chart 1 shows the sucrose gradient fractionation of the nuclei of AKR cells exposed to [3H]MC forvarying periods (4, 24, 48, and 72 hr). The distribution ofDNA represents a typical profile. This distribution does vary±5%among the gradients. The distribution of radioactivityin the gradients is similar when using nuclei from cells

exposed to [3H]MC for 4, 24, and 48 hr, with a markedlocalization of radioactivity in Fraction I. However, atthe 72-hr exposure, the radioactivity associated with all fractions isgreatly reduced. Chart 2 shows the same results plotted ascpm/mg of DNA. On this basis an even more dramaticlocalization in Fraction I is observed, with a maximal binding occurring at 24 hr of exposure. The localization oftnitium in these experiments represents total binding, i.e.,

both organic solvent-extractable and nonextractable radioactivity. Table i shows the relative distribution of the radioactivity in the 3 fractions during the various periods ofexposure. The marked localization in Fraction I occurs atthe 4-, 24-, and 48-hr exposure periods but is absent in the72-hr exposure.

Binding of Other Compounds to the Nuclear Subfraction. The 24-hr exposure period was then selected for fur

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Chart 2. Binding of [3H]MC to the nuclear fractions per mass DNA. Theseexperiments were performed as described in the legend of Chart 1. Thepooled regions of the gradients representing Fraction I (•),Fraction II (U),and Fraction III ( 0) were assayed for radioactivity as described In “Materialsand Methods―and elsewhere (29). The cpm/mg DNA was calculated from thecpm/0.2-mI aliquot and the @gDNA/0.2 ml aliquot of each fraction.

Table 1

a Each value represents the percentage of the total radioactivitymeasured in each region of the gradient designated in Chart 1 as I,II, and Ill (pellet).

then study, since maximal levels of binding occur at thisperiod . Chart 3 shows the binding of the strong carcinogens[3H]BP and [3HJDB(a,h)A, as well as of the weak carcinogen[3H]DB(a,c)A, to the nuclear subtractions after a 24-hr exposure. Whereas the strongly carcinogenic compounds showan extensive binding to Fraction I, the weak carcinogendisplays little binding. These results support those reportedpreviously for Fraction I binding of DB(a,h)A and DB(a,c)Aat the 4-hr exposure period (29). Labeled steroid hormones(progesterone and hydmocortisone) also show a low binding after 24 hr of exposure to all fractions with no Fraction Ilocalization (unpublished observation). Table 1 also showsthe relative distribution of the radioactivity for each of the

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Chart 3. Binding of other PAH to nuclear fractions after a 24-hr exposureto cells. The exposure to cells and analysis of the nuclear binding of [‘H)BP(•),[3H]DB(a,h)A(U),and[3H]DB(a,c)A(0) arethe sameasdescribedin thelegend of Chart 1 for [3HJMC,except that the concentration of these cornpounds in the cell culture bottles was 1.0 pg/mI of media.

FRACTION NUMBERChart 1. Binding of [3H]MC to nuclear fractions during increased periods

of exposure to cells. AKR-2B cells were grown in the presence of I3HIMC(0.4pg/mI, 670 Ci/mol) for 4 hr (C), 24 hr (U), 48 hr (0), and 72 hr (0). Afterharvesting of the cells by trypsinization and washing with culture medium,nuclei were isolated, mechanically sheared, and centrifuged on a sucrosegradient as described elsewhere (29, 42). The cpm/0.2-mI aliquot of eachfraction is shown (—), and the DNA per fraction (*) was determined asdescribed In “Materialsand Methods.―The concentration of sucrose perfraction (- - -) was estimated with a refractometer. Fractions I and II designate the regions of the gradient with fractions that were pooled as describedpreviously (42). Fraction III represents the pellet in the gradient tubes. Thegradients were fractionated into 1.8-mI fractions; the pellet was resuspendedin 6 ml of 0.01 N Tnis buffer, pH 8.0.

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Nuclear Binding of MC Metabotites

various PAH compounds in the nuclear fractions. All thePAH, including the DB(a,c)A, display similar distribution inthe various nuclear fractions. Consequently, only the levelof nuclear binding and not the relative distribution amongthe fractions appears to differ between the potent and weakcarcinogens after 24 hr'of exposure. Our previous paper(29) reported that the level of nuclear binding and the nelative distribution among the fractions differed between thepotent and weak carcinogens after 4 hr of exposure to cells.We now observe some localization of this weak cancinogenic compound in Fraction I, although the level of bindingis still much lower than that of the carcinogenic compounds.

Level of Nonextractable Radioactivity. The amount ofradioactivity that is not extractable in organic solvents duning the various periods of exposure of cells to [3HJMCwasthen analyzed. The methods to assess this extractabilitywere Method I (benzene extracts of aqueous solutions ofnuclear subtractions) and Method II (toluene extraction ofpreparations of the fractions dried on Millipore filters) andare described in “Materialsand Methods.―The levels ofDNA and protein remain unchanged during the extractionswith Methods I and II. Thus, Method II appears to be supenor since it removes more radioactivity from the fractions.In Method I, the chromatin condenses in the organic solventand probably traps some of the radioactivity, whereas inMethod II the chromatin cannot condense since it is “fixed―on the membrane filters. This aspect, combined with thegreater ratio of the volumes of extracting organic solvent tochromatin in Method II compared to Method I, explains themore thorough extraction of radioactivity by Method II. In aseries of experiments, the 3rd extraction of Fractions I, II, orIll in either Method I on II consistently removed only slightlymore radioactivity than did the 2nd extraction (see Chart 4).Thus, 3 extractions were subsequently applied to each ofthe methods. Chart 5 shows the percentage of nonextractable radioactivity (covalent binding) in the nuclear subfnactions during increasing periods of exposure of cells to[3H]MC. At 4 hr of exposure, 10 to 20% of the radioactivity isnot extractable. As the period of exposure of the cells to[3HJMC increases, the percentage of nonextractable radioactivity increases up to a level of 50 to 70% (at the 72-hr

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Chart 5. Percentage of total radioactivity not extractable by organic solvents. Nuclei cells exposed to [‘H]MCfor increasing exposure periods werefractionated to obtain Fraction I (A), Fraction II (B), and Fraction Ill (C). Thesefractions were then extracted with organic solvents by Method I (U) usingbenzene or by Method II (—) using toluene as described in “MaterialsandMethods.―A and t@,duplicate experiments using the Method II extraction.The percentage of nonextractable cpm was then calculated.

period). Chart 6 shows the level of this residual radioactivity(using Method II) in each fraction for each exposure period.This nonextractable radioactivity in each of the fractionsshows a pattern similar to that in Chart 2, which representsthe combined extractable and nonextractable radioactivity.In both instances, the binding per mass DNA in Fraction Idisplays a higher level of binding than that in Fractions IIand Ill, and the 24-hr exposure period displays higher binding pen mass DNA than any of the other exposure periods.

Molecular Species Bound to the Various Fractions. Fordetermination of the extent of metabolites of [3HJMCassociated with the fractions, each fraction was extracted withbenzene as described in “Materials and Methods―(MethodI), and the extracts were lyophilized and analyzed by HPLC.Chart 7 shows a typical elution pattern of metabolites of MCusing the reverse phase system described in “Materials and

Methods.―This reverse phase system represents the best ofmany investigated in this laboratory using the Waters Instrument and Waters C1, @Bondapak column, with normal and

reverse phases, a variety of solvents, and linear and curvedgradients. A reverse phase system with good resolvingpower for separating BP and its metabolites has previouslybeen reported by Selkirk et at. (34, 35) using a Dupontinstrument and a Dupont ODSPermaphase column. Chart 8shows the elution of the extractable radioactive components from Fraction I of cells exposed to [3H]MC for 4, 24,and 48 hr. Since similar patterns were obtained for Fnactions 2 and 3, profiles of their radioactive components arenot shown. Chart 9 presents the percentage of the totalextractable radioactivity in Fractions I, II, and III that represents metabolites of [3HIMC. The data were calculated fromthe data of Chart 8 for Fraction I and from similar expemi

1@0HOURS of EXPOSURE

Chart 6. Level of nonextractable radioactivity per mass DNA in the nuclearfraction. The experiment was essentially the same as that described in thelegend of Chart 2 except that radioactivity was extracted by the toluenemethod (Method II) as described in “Materialsand Methods.―

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Chart 4. Extraction of radioactivity from the nuclear subfraction by Methods I and II. Nuclear subfractions from cells exposed to [‘HIMCfor 24 hr weresubjected to 3 sequential extractions of benzene (Method I) (U) or toluene(Method II) (•)as described in “Materialsand Methods.―After each extraction, lOO-pi aliquots of each extract were counted, and the total cpm in thewhole extract were calculated . The radloactivities in all 3 were added , andsequential extractions represented the total extractable radioactivity. Thepercentage of the total radioactivity distributed in each of the 3 extractionswas then calculated. The means and ranges of the values obtained from 9different samples are shown. Similar results were obtained for Fractions I, II,or III from cells exposed to the [3HJMCfor the various periods.

MAY 1977 1493

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0 40 80 120

T. C. Spetsberg et at.

this type of binding and the extent of metabolism that hasoccurred.

Effects of Inhibiting PAH Metabolism on the NuclearBinding. It metabolism of MC is essential for the noneXtractable binding to the fractions, any inhbition of this metabolism should reduce this strong binding. For study of thisaspect, a predetermined dose of the flavonoid, BF, an inhibitor of amylhydrocarbon hydmoxylases (1, 9, 10, 35, 45), wasadded to the media together with the [3H]MC, and its abilityto inhibit metabolism of [3H]MC was assessed. Chart 11shows the effects of BF on the level and types of metabolitesextracted from Fraction I of cells exposed for 48 hr to[3H]MC. The inhibitor does not cause a reduction in thenumber of species of metabolites, but, as shown in Charts 9and ii , the BF markedly reduces the extent of metabolism.Similar effects of BF have been reported in studies with ratmicrosomal preparations in vitro by Selkirk et a!. (35) and inhamster embryo cells in culture by Baird and Diamond (1).After 48 hr of exposure of the cells to [3H]MC, approximately80% of the extractable radioactivity from Fraction I mepresents metabolites of MC, whereas only 19% represents metabolites of MC when BF is included. Analysis of whole-cellradioactivitydemonstratesa similarinhibitionof metabolism by BF. Consequently, the selected dose of BF waseffective in blocking much of the metabolism of [3HJMC inthe cells.

As shown in Chart i2A, the level of nonextractable radioactivity in each of the fractions after a 48-hr exposure to[3H]MC is greatly reduced when BF is present. Similar stud

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Chart 7. HPLC of MC and its metabolites. HPLC was run as described in.. Materials and Methods― with a Waters C,, u Bondapak Column. About 1 @.og

each of MC, cis-11,12-diol, 11,12-quinone, dialdehyde, 11-OH, and 11,12-epoxide was applied separately as well as together, and their elutionwas monitored by a spectrophotometer at 2540 A. The photometer signalswere recorded by a Hewlitt-Packand 3380A recording integrator as describedin “Materialsand Methods.―Repeat nuns displayed elution volumes thatvaried ±0.5ml for each of the standards. The cis-11,12-diol and 11,12-epoxide standards supplied by Dr. Sims eluted at the same volume as thecostandards supplied by the National Cancer Institute.

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Chart 8. HPLC of the benzene-extractable radioactivity from Fraction I ofcells exposed to [3HIMC for varying periods. The benzene extraction ofFraction I as well as HPLC analysis are described in “Materialsand Methods.―The patterns represent 4-hr exposure (A), 24-hr exposure (B), and 48-hrexposure (C). The elution volumes of the standards obtained from Chart 7 areindicated.

ments for Fractions II and Ill. Since the nadioactivities associated with the fractions from the 72-hr experiments werelow, larger amounts of cells were used in the extractions toincrease the level of radioactivity. Chart 10 more clearlyshows the relationship between the metabolites in the extractablefractionand theextentofnonextractableradioactivity. This relationship is linear in a semiloganithmic plot,which could be a result of induction of drug-metabolizingenzyme activities or of a 2-step activation mechanism forthe covalent chromatin binding . Since the major extractablemetabolites associated with each of the fractions elute atperiods similar to those of our standards, the 11-OH, ii ,12-dihydroxy,11,12-quinone,and possiblythe 11,12-epoxide derivatives, it is possible that they represent these metabolites. However, the exact identification of these metabolites must await further analysis. Also, the exact nature ofthe nonextractable molecular species is not known. In anycase, these results do show that, the longer the period ofexposure of the cells to the [3HJMC,the greater the amountof metabolites associated with the fractions. These resultsalso suggest that the metabolism of the parent compoundmay be required for the covalent binding to the chrorno

somal material, since there is a direct correlation between

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HOURSof EXPOSUREChart 9. Percentage of the extractable radioactivity of the nuclear frac

tions representing metabolites of MC. The HPLC data from Chart 7 as well assimilar data for cells exposed for 72 hr were used to calculate the percentageof metabolites. The radioactivity eluting with the metabolites of MC and thetotal radioactivity eluted from the columns were used to calculate the percentage of the total extractable radioactivity representing the metabolites ofMC. These data are plotted versus the hours of exposure of [‘H)MCto thecells with Fraction I (•),Fraction II (U), and Fraction III ( 0). - - -, fractionsfrom cells exposed to [3H]MC and BF (3 @g/ml).

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versusthe percentageof metabolites.Thesedata weretakenfrom Charts5and 9 and plotted on a semiloganithmic scale.

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Nuclear Binding of MC Metabolites

interaction with cellular macromolecules other than DNA.Since the DNA within the cell is covered with proteins andRNA, binding of electrophilic, ultimate carcinogens to theselatter macromolecules is highly probable. There have beenreports that carcinogenic PAH bind to whole chromatin aswell as to nuclear histone and acidic proteins (5, 19, 24, 30).Moreover, chemical carcinogens have been shown in someinstances to bind to chromosomal proteins and RNA to agreater extent than to DNA (13, 17, 31). These results arecertainly intriguing, since these chromosomal componentsare suspected of involvement in gene regulation (38). Unfortunately, none of the many hydrocarbon-binding sites incells have been shown to be specific only for carcinogeniccompounds.

The studies presented here extend those reported previously (29, 42) in demonstrating a specific, high-affinity localization of the carcinogenic PAH to a specific nuclearsubfraction. The extensive binding to Fraction I after a 24-hrexposure period demonstrates a specificity with respect tocarcinogenic PAH [MC, BP, and DB(a,h)A], while the weakor noncarcinogenic compounds [steroids and DB(a,c)Aidisplay less binding. The reasons for this specificity areunclear, especially since it is observed after 4 hr of exposurewhenein little metabolism ofthe PAH hasoccurred. Themain differenceinFractionIbindingbetweentheshort(4-hr) and long (24- to 72-hr) exposures to the labeled carcinogens is an increased proportion of nonextractable radioactivity of the latter, which in turn appears to be dependent onmetabolism of the parent compounds. Whether this nonextractable radioactivity represents covalent binding andwhether this type of binding is a requirement for malignanttransformation of cells remain to be determined. Severalspecies of metabolites are extracted from each of the nuclear fractions as assessed by HPLC. The number and natune of the species in the nonextractable radioactivity is notknown. Whatever the multiplicity of species, we calculateabout 50 x 106molecules/cell associated with Fraction I, ofwhich only 10 million are tightly bound. Surely a heterogeneity of both the bound species of PAH and the chromatincomponents (bound by these species) exists.

There appears to be a relationship between the extent ofmetabolism (at least the proportion of metabolites in thefractions) and the degree of nonextractable radioactivity(covalent binding) in the chromatin fractions. BE, an inhibitor of drug metabolism, prevents this covalent binding toour subnuclear fractions. BE has previously been reportedto block BP from covalently binding to DNA (1, 35). Thisrelationship between metabolism and nonextractable radioactivity (covalent binding) is based on the length of incubation of the cells with [3H]MC. That this relationship betweenmetabolism and covalent binding is linear in a semilogarithmic plot suggests that there may be a 2-step activation

involved in the covalent binding of the [3H]MC to the nuclearsubfractions. A similar relationship and interpretation wasreported by Baird and Diamond (1) with [3H]BP in hamsterembryo cells. A 2-step activation mechanism for DNA binding has also been suggested with evidence for BP (3, 22).The diol-epoxides of benz(a)anthnacene (2, 39) and BP (36,44) found complexed to DNA also support this theory.

We are presently attempting to assess more thoroughlythe specificity for Fraction I localization using a variety of

0 80 160

40

20

0

RETENTIONVOLUME(mI)

Chart 11. HPLC of the extractable radioactivity from Fraction I from control cells (A) and cells treated with BF (B). The treatment of cells, isolation ofNuclear Fraction I, and extractions of the radioactivity are described in“Materialsand Methods.―A , patterns from Fraction I from cells treated with[3H]MC alone (0.4 @g/mlof media) for 48 hr; B, patterns from Fraction I fromcells treated with 0.4 @&gof [3H]MC and 3 @gof BF per ml of media for thesame period.

Ui-a Ui@ -i804

OC@ 24 48@ 24 48

HOURSof EXPOSURE HOURS of EXPOSUREChart 12. Effects of BF on the percentage of nonextractable radioactivity

with [3H]MC and [3H]BP. Cells were incubated for increasing periods with[3H]MC (0.4 @g/ml)(A) and with [3Hjbenzo(a)pyrene (1.0 @g/ml)(B). Thepercentage of nonextractable radioactivity with each carcinogen in each ofthe fractions was determined, and the values were designated (—). 5omecells were incubated with BF (3.0 @g/ml)for 48 hr (A) and for 24 hr (B). - - -,binding values in expenimentswith BF. The nucleanfractions l(U), II (A), andIII ( 0) were obtained as described elsewhere (42).

ies have been performed with [3H]BP (Chart 12B), whereinthe nonextractable radioactivity in each fraction from cellsexposed to [3H]BP is similarly reduced when BF is includedin the incubation. Consequently, it appears that the metabolism of the potent carcinogenic compounds is essential forcovalent binding of these compounds to the nuclear fractions. The extractable radioactivity (noncovalent binding)does not require metabolism, inasmuch as short exposureperiods resL ,t in the marked association of the parent[3H]MC to the suonuclear fractions (especially to Fraction I),and theBF does notreducethistypeofbinding.

DISCUSSION

There have been numerous studies on binding of PAH toDNA, RNA, and proteins of cellsin vivo (i3, 23), in vitro (31),and in tissue culture model systems (12, i6, 25). Moststudies on PAH binding within the cell have been concernedwith covalent binding to DNA (17, 28), since a correlationbetween the capability of a compound to bind covalently toDNA and its carcinogenic activity has been reported (6, ii).However, evidence contradictory to this correlation hasarisen (15, 25). Chemical carcinogens could act through an

MAY 1977 1495

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T. C. Spelsberg et at.

bon in Chinese HamsterCellsand Human Leukocytesin Wtro. Hereditas,59: 120-141, 1968.

21. Kato, R., Bruze, M., and Tegner, V. Chromosome Breakage Induced invivo by a Carcinogenic Hydrocarbon in Bone Marrow Cells of theChinese Hamster. Heneditas, 61: 1-8, 1969.King, H. W. S., Thompson, M. H., and Brookes, P. The Benzo(a)pyreneDeoxynibonucleoside Products Isolated from DNA after Metabolism ofBenzo(a)pyrene by Rat Liver Microsomes in the Presence of DNA. Cancer Res., 35: 1263—1269,1975.

23. Kinoshita, N., and Gelboin, H. V. Aryl Hydrocarbon Hydroxylase andPolycyclic Hydrocarbon Tumonigenesis: Effect of the Enzyme Inhibitor7,8-Benzoflavone on Tumonigenesis and Macromolecule Binding. Proc.NatI. Acad. Sci. U. 5., 69: 824—828,1972.

24. Kodama, M., Tagashina, V., and Nagala, C. Interaction of AromaticHydrocarbons with Deoxynibonucleoprotein in Vitro . J. Biochem ., 65:81-86, 1968.

25. Kuroki, T., and Heidelberger, C. The Binding of Polycyclic AromaticHydrocarbons to the DNA, RNA, and Proteins of Transformable Cells inCulture. Cancer Res., 31: 2168—2176,1971.

26. Loeb, L. A., and Gelboin, H. V. Methylcholanthrene-Induced Changes inRat Liver Nuclear RNA. Proc. NatI. Acad. Sci. U. 5., 52: 1219—1226,1964.

27. Madix, J. C., and Bresnick, E. Increased Efficacy of Liver Chromatin as aTemplate for RNA Synthesis after Administration of 3-Methylcholanthrene. Biochem. Biophsy. Res. Commun., 28: @5—452,1967.

28. Miller, J. A. Carcinogenesis by Chemicals: An Overview. Cancer Res., 30:559-576, 1970.

29. Moses, H. L., Webster, R. A., Martin, G. D., and Spelsbeng, T. C. Bindingof Polycyclic Aromatic Hydrocarbons to Transcniptionally Active NuclearSubfnactions of AKR Mouse Embryo Cells. Cancer Res., 36: 2905-2910,1976.

30. O'Brien, R. L., Stanton, A., and Craig, R. L. Chromatin Binding ofBenzo(a)pyrene and 20-Methylcholanthrene. Biochim. Biophys. Acta,186:414-717, 1969.

31. Pezzuto, J. M., Lea, M. A., and Yang, C. S. Binding of MetabolicallyActivated Benzo(a)pyrene to Nuclear Macromolecules. Cancer Res., 36:3647-3653, 1976.

32. Reiman, H. M., Branum, E. L., and Moses, H. L. Effects of ChemicalCarcinogenes and C-Type RNA Viruses on Transformation of MouseEmbryo Cells. J. Cell Biol., 67: 538a, 1975.

33. Rowe, W. P., Hartley, J. W. , Lander, M. R., Pugh, W. E. , and Teich, N.Noninfectious AKR Mouse Embryo Cell Lines in Which Each Cell Has theCapacity to Be Activated to Produce Infectious Munine Leukemia Virus.Virology, 46: 866-876, 1971.

34. Selkirk, J. K., Croy, R. G., and Gelboin, H. V. Benzo[ajPyrene Metabolites: Efficient and Rapid Separation by High Pressure Liquid Chromatography. Science, 184: 169-170, 1974.

35. Selkirk, J. K., Croy, R. G., Roller, P. P., and Gelboin, H. V. High PressureLiquid Chromatographic Analysis of Benzo[ajpyrene Metabolism andCovalent Binding and the Mechanism of Action of 7,8-Benzoflavone and1,2-Epoxy-3,3,3-tnichlonopropane. Cancer Res. , 34: 3474-3780, 1974.

36. Sims, P., Grover, P. L., Swaisland, A., Pal, K., and Hewer, A. MetabolicActivation of Benzo[ajpyrene Proceeds by A Diol-epoxide. Nature, 252:326-328, 1974.

37. Spelsbeng, T. C. The Role of Nuclear Acidic Proteins in Binding SteroidHormones. In: I. L. Cameron and J. R. Jeter, Jr. (eds.), Acidic Proteins ofthe Nucleus,pp. 249-296.NewYork: AcademicPress,Inc., 1974.

38. Stein, G. S., Spelsbeng, T. C., and Kleinsmith, L. J. Nonhistone Chromosomal Proteins and Gene Regulation. Science, 183: 817—824,1974.

39. Swaisland, A. J., Hewer, A., Pal, K., Keysell, G. R., Booth, J., Grover, P.L., and Sims, P. Polycyclic Hydrocarbon Epoxides: The Involvement of8,9-Dihydro-8,9-dihydroxybenz[a]anthracene 10,11-Oxide in Reactionswith the DNA of Benz[a]anthracene-treated Hamster Embryo Cells. Federation European Biochem. Soc. Letters, 47: 34-38, 1974.

40. Teich, N., Lowry, D. R., Hartley, J. W., and Rowe, W. P. Studies of theMechanism of Induction of Infectious Munine Leukemia Virus from AKRMouse Embryo Cell Lines by 5-Lidodeoxyunidine and 5-Bromodeoxyuridine. Virology, 51: 163-173, 1973.

41. Turkington, R. W. Changes in Hybridizable Nuclear RNA during theNeoplastic Development of Mouse Mammary Cells. Cancer Res., 31:427-432, 1971.

42. Webster, R. A., Moses, H. L., and Spelsbeng, T. C. Separation andCharacterization of Transcniptionally Active and Inactive NuclearSubfractions of AKR Mouse Embryo Cells. Cancer Res., 36: 2896-2904,1976.

43. Weinstein, B., Yamaguchi, N. , Gebert, R., and Kaighn, M. E. Use ofEpithelial Cell Cultures for Studies on the Mechanism of Transformationby Chemical Carcinogens. In Vitro, 11: 130—141, 1975.

44. Weinstein, I. B., Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Harvey,R. G., Harris, C., Autrup, H., Kasai, H., and Nakanishi, K. Benzo[ajpyneneDiol Epoxides as Intermediates in Nucleic Acid Binding in Vitro and inVivo. Science, 193: 592-595, 1976.

45. Wiebel, F. J., Leutz, J. C., Diamond, L., and Gelboin, H. V. Aryl Hydnocarbon (Benzo[aJ-pyrene) Hydroxylase in Microsomes from Rat and Organic 5olvents. Arch. Biochem. Biophys., 144: 78-86, 1971.

1496 CANCER RESEARCH VOL. 37

additional radioactive compounds in competitive bindingassays. The identification of these chromatin componentsbound, as well as the species of the radioactive ligandbound, is planned. The role of DNA replication in the extentof binding of [3H]MC and [3H]BP to Fraction I over various 22.periods of cell exposure is presently being examined, sincethe maximal binding measured after 24 hr of exposure maybe dependent on this process.

ACKNOWLEDGMENTS

We thank Dr. W. P. Rowe and Dr. N. Teich (National Institute of Allergy andInfectious Diseases) for supplying seed stocks of the AKR mouse embryo cellline and Dr. P. Sims (Institute of Cancer Research, Royal Cancer Hospital,ChesterBeattyResearchInstitute)for hisgift of severalof thestandardPAH.We also thank Mn. Brad Syverson, Ms. Barbara Gosse, and Ms. Mary E.Volkenant for their excellent technical assistance.

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1977;37:1490-1496. Cancer Res   Thomas C. Spelsberg, Thomas H. Zytkovicz and Harold L. Moses  Embryo CellsHydrocarbons to Nuclear Subfractions of Cultured AKR Mouse Effects of Metabolism on the Binding of Polycyclic

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