(U-22), Fondation Curie-Institut du Radium, Bâtiment110 ¡F.2.). 91405 Orsay, France
ABSTRACT
Dibenzo(a,e)fluoranthene (DBF), a highly carcinogenicpolycyclic hydrocarbon without an apparent K-region,binds covalently to DNA, transfer RNA, and polyribonu-cleotides when incubated with hepatic microsomal fractions under standard conditions. Optimal binding conditions for [3H]DBF were established.
Methylcholanthrene-pretreated mouse liver micro-somes induced a higher level of binding of [ H]DBF toDMA than did similarly induced rat liver microsomes. 7,8-Benzoflavone strongly inhibited the binding of this polycyclic aromatic hydrocarbon to DMA, while cyclohexeneoxide and trichloropropene oxide had an enhancing effectwhen used in the presence of rat liver microsomes. Anunexpected inhibitory effect was observed with cyclohexene oxide in mouse liver microsome-enriched medium.
[3H]DBF bound twice as much to denatured as to nativeDMA. Incubation of [3H]DBF in the presence of liver microsomes and polyribonucleotides (polyadenylate, polyuri-dylate, polyguanylate, and polyinosinate) indicated thatbinding occurs mainly with guanine. Binding of [ 'H]DBF to
DMA of various origins was found to be directly proportional to the amount of GC pairs. Preliminary resultsindicate a covalent bond between DBF and nucleic acids.
INTRODUCTION
Lacassagne ef al. (23) demonstrated the powerful carci-nogenicity of DBF3 in mice. This PAH belongs to the familyof benzo- and dibenzofluoranthenes, carcinogenic pollutants as common as BP. They are found in large quantitiesin tars, e.g., cigarettes (40); exhausts of urban areas andcombustion engines; road dusts as well as soil and sediments close to sea coasts, lake fronts and river banks; oiltars; creosote oil; and smoked foods (16).
The carcinogenicity of a few benzofluoranthenes onmouse skin was studied by Wynder ef a/. (45), and theelectronic properties and sarcomagenicity of several benzo-
and dibenzofluoranthenes were analyzed by Lacassagne efal. (24).
For a long time DBF was confused chemically withdibenzo(a,/)pyrene (28) (Chart 1). Subsequently, both hydrocarbons were unambiguously prepared by total synthesis (7, 29, 42). Tested again under similar conditions, theyboth showed high sarcomagenicity (26)."
A prominent difference in electronic structure distinguishes the 2 PAH's. To be carcinogenic all dibenzopyrenesmust apparently have a K-region (22, 23, 27). Dibenzo-(e,/)pyrene, which is completely devoid of such a region, istotally inactive (25). However, DBF, which has no apparentK-region. is a strong carcinogen.
The hypothesis that K-region epoxides represent theultimate carcinogenic metabolites (4, 37) is now beingquestioned (3, 8, 15, 20, 31, 38) despite the fact that the K-region epoxides are highly mutagenic toward bacteria (2)and display a potent transforming activity toward cells inculture (14).
The strongest evidence against this theory is the findingthat all K-region oxides obtained by synthesis and tested inmice by several authors have low or no carcinogenic activity(5, 30, 32). On the other hand, the recent data on mutagen-icity, binding to nucleic acid, and carcinogenicity of different metabolites of BP point to the products of secondaryoxidative metabolism, such as vicinal epoxide dihydrodiolsof non-K-region rings as the most probable ultimate carcinogenic metabolites (8, 19, 38).
These observations led us to compare the metabolism of2 structurally different PAH's, one without any apparent K-
region (DBF) and the other containing a typical K-region(i.e., BP). Hopefully, such a comparison will contribute to aclearer definition of the molecular structures of PAH-gen-erating carcinogenic metabolites.
A series of DBF derivatives was synthesized and testedfor their carcinogenic activities on XVIInc/Z mice.4 In addi
tion the metabolism of DBF was investigated. When incubated in the presence of a NADPH-dependent microsomalsystem (11), DBF is transformed into some 20 metabolitesthat are currently being identified.5
Many studies have dealt with the identification of theinteraction between PAH and cellular components; theyprovide strong evidence for covalent binding of PAH to
4 F. Zajdela, O. Perin-Roussel. and M. Croisy-Delcey. The CarcinogenicActivities of Benzo- and Dibenzofluoranthenes, manuscript in preparation
50. Perin-Roussel. M. Croisy-Delcey, J. Mispelter. S. Saguem. F. Zajdela.B. Ekert, P. Jacquignon, J.-M. Lhoste. and B. Muel. Metabolism of PolycyclicAromatic Hydrocarbons: The Metabolism of Dibenzo(a,e)Fluoranthene byLiver Homogenates, manuscript in preparation.
DNA, RNA, and cellular proteins both in vivo and in vitro (6,17, 21, 43). PAH metabolite binding to DNA being widelyrecognized as an important and perhaps major event in theprocess of neoplastic transformation has led us to investigate the binding of DBF to a variety of nucleic acids in thepresence of mouse and rat liver microsomes.
MATERIALS AND METHODS
Preparation of Microsomes. Mouse liver microsomeswere prepared from XVIInc/Z mice. This highly hybridizedstrain belongs to the category of mouse strains responsiveto aromatic hydrocarbon induction (33).
Induction of AHH was brought about by i.p. injection ofMC, 4 mg/100 g, 48 hr before killing. It is generally admittedthat MC is a good inducer of AHH involved in the productionof ultimate PAH metabolites (33).
Rat liver microsomes were prepared from our strain ofWistar rats which were given injections of MC, 1 mg/100 g,48 hr before killing. Relatively weak doses of MC were usedto avoid the excess fatty degeneration of the hepatocytesaffected by the toxic MC metabolites that often hampersmicrosome preparations. For the same reason we avoidedfasting the mice before killing.
Mouse and rat liver microsomes were prepared accordingto the techniques described by Garner ef al. (10) and Amesef al. (1). The microsomes were suspended in Tris-sucrosebuffer at a concentration of 12 mg protein per ml, and 1-mlaliquots were stored in liquid nitrogen. Protein concentration was determined by the biuret reaction (12) with bovineserum albumin as standard. Each stage of the procedurewas checked microscopically.
Chemicals and Radioisotopes. DBF was synthesizedaccording to the technique described by Lavit-Lamy ef al.(28, 29). The hydrocarbon was again purified chromato-graphically on 60 Merck thin-layer Kieselgel (Merck AG,Darmstadt, West Germany) in a cyclohexane/benzene solvent (80/20).
[3H]DBF (prepared by Mr. Audinot, Isotope Department ofC.E.A., Saclay, France) was obtained by isotopie exchangein TzO; 120 mg of DBF in 2 ml of T20 containing 70 Citritium were shaken during 48 hr at 110°in the presence ofPtO2 reduced by 10 ml of tritium gas (25 Ci). A 3H nuclear
magnetic resonance spectrum (64 mHz) (Bruker W60 spectrometer, Spectrospin S.A. France, Rue de l'Industrie, 67
Wissembourg, France) indicated a uniform distribution oftritium in [3H]DBF.
[3H]DBF was again purified by 2 consecutive thin-layer
chromatographies, the first using cyclohexane/benzene(80/20) as solvent and the second using cyclohexane/diox-ane (80/20).
The labeled compound (910 mCi/mol) was then placed inacetone at a concentration of 250 /¿g/mland stored in thecold and dark.
DNA samples from different animal and bacterial specieswere purchased from Sigma Chemical Co. (St. Louis, Mo.).They were placed in 0.01 M NaCI at a concentration of 3mg/ml. DNA solutions were denatured by heating at 100°for 4 min followed by immediate cooling to 0°.
Polyribonucleotides (Sigma) were prepared as anaqueous solution at a concentration of 15 HIM and estimated spectrophotometrically, after alkaline hydrolysis with0.3 M KOH for 18 hr a 37°.MRE 600 Escherichia coli tRNA(Boehringer-Mannheim GmbH, Munich, West Germany) inaqueous solution was used at a concentration of 3 mg/ml.
Determination of Microsome-mediated Binding ofPHJDBF to Nucleic Acids. The assay system used formicrosome-mediated binding of [3H]DBF to nucleic acids
was based on the system originally described by Gelboin(26). Each tube contained, in a total volume of 1 ml, 50/¿molof Tris-HCI (pH 7.5), 0.36 /¿molof NADPH, 3 Mmol ofMgCU, 0.1 ml of the microsomal preparation (containing1.2 mg proteins), and 15 /xl of an acetone solution of[3H]DBF at 250 ^g/m\ added just before incubation, i.e.,3.75 /ng [3H]DBF. Quantities of nucleic acid and other
ingredients are indicated in the chart and table legends.Reaction mixtures, prepared at 0°,were incubated at 37°
and slightly stirred throughout incubation. To each tubewas added 0.1 ml 10% sodium dodecyl sulfate, and theassay was left for 5 min at room temperature before additionof 1 ml of water-saturated phenol. The tubes were periodically shaken with a Vortex at 20°during 20 min. Sampleswere centrifuged in a low-speed centrifuge to separate theaqueous and phenolic phases completely. The aqueousphase was removed with a Pasteur pipet and extracted 4times with 1 ml ether, 4 times with 1 ml ethyl acetate, andagain once with 1 ml ether. Excess residual ether waseliminated by a light air current. A 0.5-ml sample was takenfrom each tube, and the DNA fraction was precipitated byaddition of either 0.5 ml 10% TCA or 1 ml ethanol and 50 /¿I20% potassium acetate. The assays were left at roomtemperature for 30 min filtered on a Whatman GF/C glassfiber filter. The TCA precipitate was rinsed twice with 3 ml5% TCA. Both types of precipitate were washed twice with3 ml ethanol and twice with 3 ml ethyl acetate. The filterswere dried under an IR lamp and covered with 10 mlscintillation liquid containing 4 g PPO and 0.1 g dimethyl-POPOP per liter toluene, after which 0.1 ml M Hyaminehydroxide in methanol was added. The mixture was thenleft for 2 hr at room temperature, and radioactivity wasdetermined in an Intertechnique scintillation counter (Inter-technique, 78370 Plaisir, France).
No significant differences were observed between those2 precipitation techniques. DNA recovery was measuredafter each test and found to be almost always total.
For tRNA and polyribonucleotide recovery, an aliquot of
the aqueous phase (0.5 ml) was precipitated with 5% TCA.The precipitate was centrifugea and hydrolyzed in 1 ml 0.6M NaOH at 37°for 18 hr. The pH of the solution was adjusted
to 7 with 1.2 ml 0.5 M HCI.and its absorbance was measuredat the maximum absorption wavelength corresponding tothe base of the polyribonucleotide under study.
RESULTS
Optimal Experimental Conditions for [3H]DBF Binding to
DMA
Determination of Incubation Time. We measured theamount of [3H]DBF binding to salmon sperm DNA (0.9 mg/assay) in an NADPH-dependent microsomal system as afunction of incubation time. Chart 2 shows that [3H]DBF
simultaneously binds to the DNA and to the endogenousmicrosomal RNA's. Binding gradually increased during the
first 30 min of incubation for both native and denaturedDNA, as well as for endogenous RNA, before reaching aplateau. The amount of DBF bound to denatured DNA washigher than that bound to native DNA.
[3H]DBF Binding as a Function of DNA Concentration.Maximum binding of [3H]DBF was achieved at 0.3 mg DNA
per assay (Chart 3). For higher concentrations the bindingslowed down for both native and denatured DNA. All theDNA was recovered in 5% TCA without noticeable loss ofsoluble material.
[3H]DBF Binding as a Function of the Quantity of Micro-somes. Maximum binding to DNA was attained when theconcentration of microsomal proteins was 1.2 mg/assay;binding of [3H]DBF to microsomal RNA gradually rose to alimiting value of about 2.4 mg protein per assay (Chart 4).Therefore we always used 1.2 mg microsomal protein perassay, a quantity that allowed maximum binding to DNA butlimited binding to microsomal RNA.
[3H]DBF Binding as a Function of Its Concentration.
l.tlm.n
Chart 2. Kinetics of [3H]DBF binding to native and denatured salmonsperm DNA. Each determination was made with 0.9 mg DNA according tothe procedures described in "Materials and Methods." •¿�.denatured DNA;
O, native DNA; A. without DNA.
150.
oo
100.
50.
0.3 O.6 0.9 1.2 mg DNAChart 3. [3H)DBF binding as a function of the concentration of salmon
sperm DNA. Incubation time for each sample was 40 min. •¿�,denatured DNA;O, native DNA. The assay conditions are as described in "Materials andMethods."
123 mg proteinsChart 4. [3H]DBF binding as a function of microsomal protein concentra
tion. Each sample was incubated for 60 min; 0.3 mg native and denaturedDNA were used for each determination. •¿�.with denatured DNA; O, withnative DNA; A. without DNA.
[3H]DBF binding experiments were carried out with native
and denatured DNA (0.3 mg/assay) to determine the minimum amount of [3H]DBF necessary to saturate the system.This value was found to be 3 /¿g/assay(Chart 5). This iscertainly an overestimation since [3H]DBF was present in
the reaction mixture as a very fine suspension and wastherefore not completely dissolved. Several control testswere carried out by adding an NADPH enzyme-regeneratingsystem (composed of glucose 6-phosphate and glucose-6-phosphate dehydrogenase) to the microsomal system. Nosignificant differences were observed as compared to ouroptimal standard conditions.
[3H]DBF Binding in the Presence of Specific Inhibitors.
Chart 5. [3H]DBFbinding as a function of its concentration. Determinationswere made with 0.3 mg native and denatured salmon sperm DNA. Incubationlasted for 40 min. •¿�.denatured DNA; O, native DNA.
The influence of certain specific inhibitors of monooxygen-ases and epoxide hydratases on [3H]DBF binding to DNA
was investigated.7,8-Benzoflavone, known to inhibit monooxygenases
(44), inhibited [3H]DBF binding to DNA in both mouse andrat MC-pretreated liver microsomal systems (Table 1).
We confirmed that TCPO, an epoxide hydratase inhibitorthat acts by reducing epoxide hydrolysis (34), increased[3H]DBF binding to DNA. This is true at weak TCPO concen
trations. In mouse liver microsome experiments, the maximum effect was obtained at a concentration of 10 //M, andin the rat liver microsome experiments, it was obtained at aconcentration of 20 /J.M. These results agree with those ofPietropaolo and Weinstein (35), who obtained a similareffect for BP binding to E. coli tRNA. However, when TCPOconcentrations were higher than 0.1 mw, we observed astrong inhibitory effect with mouse liver microsomes butnot with rat microsomal system.
Similar experiments were carried out with CHO as epoxide hydratase inhibitor.
As expected, CHO increased [3H]DBF binding to DNA in
experiments with rat liver microsomes. Contrariwise, in thepresence of mouse liver microsomes, CHO inhibited thebinding by 20% at concentrations between 10 ¿¿Mand 0.1mw; for higher concentrations this effect decreased (Table1). The discrepancy of these results cannot yet be explained.
[ 'HJDBF Binding to Different Polyribonucleotide and Poly-
deoxyribonucleotide Chains
The affinity for [3H]DBF of various purines and pyrimidinein the DMA-binding reaction was investigated by incubatingdifferent single-sequence polyribonucleotide chains withthe PAH under optimal experimental conditions. Experiments with polyuridylate were inconclusive because ofextreme sensitivity of this polyribonucleotide to endogenous nucleases, which hydrolyze it very fast into productsnot precipitable by 5% TCA. Polyadenylate, polycytidylate,and polyinosinate, despite repeated experiments, did notreact with [3H]DBF, nor did double-stranded polynucleo-tides (double-stranded copolymer of polyadenylate and
polyuridylate; double-stranded copolymer of polycytidylate
and polyinosinate). On the other hand, poly(G) showedstrong affinity to [3H]DBF (Chart 6). The extent of [3H]DBF
binding increased progressively as a function of poly(G)amount per assay up to 0.3 mg. For higher concentrationsthe curve leveled off to a plateau, indicating that a limitingquantity of bound [3H]DBF had been reached. This binding
[566 pmol/mg poly(G)] proved approximately twice as highthan for denatured DNA (265 pmol/mg) and 3.7 times thatfor native salmon sperm DNA (150 pmol/mg).
These results strongly suggest that guanine is the mainsite of [3H]DBF binding in the different nucleic acids. Toverify this hypothesis, we carried out [3H]DBF binding
experiments using native or denatured DNA or tRNA of
Table 1Effect of several inhibitors on [3H]DBF binding to denatured
salmon sperm DNAIn these experiments the DNA concentration was 0.6 mg/assay
and the incubation time was 40 min. Inhibitors dissolved in acetonewere added at the final concentration indicated. The assay conditions were the same as indicated in "Materials and Methods."
various origins with proportions of GC pairs ranging from31 to 70%. We carefully checked that all of the DMA wasrecovered. The results of these experiments show that theextent of binding is dependent on the proportion of GCpairs in the native as well as in the denatured DNA's (Table
2). In addition, current experiments to establish the chemical nature of the DBF-nucleic acid bond showed a strongresistance to hydrolysis. This bond is resistant to heating at95°for 45 min, at both pH 7.0, and 12.2, nor does TCA affectsuch binding (see "Materials and Methods"). These prelim
inary results suggest the covalent nature of the DBF-nucleicacid bond^.
DISCUSSION
DBF binds to DNA, tRNA, and polyribonucleotides whenincubated with MG-pretreated rat or mouse liver micro-somes and the cofactors necessary for microsomal mixed-function oxidase activity. The binding reached an optimalvalue after 30 min incubation when the medium contained1.2 mg of microsomal proteins, 0.3 mg of DNA, and 3 /¿gof[3H]DBF. Denatured DNA binds more [3H]DBF than native
DNA.As expected, 7,8-benzoflavone, a specific inhibitor of
AHH, strongly reduced [3H]DBF binding to DNA in both
mouse and rat microsomal systems. This suggests that themetabolites responsible for binding could be an epoxide,as seems to be the case with BP. This conclusion still mustbe confirmed since this compound can either inhibit oractivate monooxygenases (39). With the epoxide hydraÃaseinhibitors, the observed effects are more complex. TCPO,when used at low concentrations (10 /¿Mto 0.1 HIM),enhances [3H]DBF binding to DNA. At higher concentra
tions we observed an inhibition with mouse microsomes(40% inhibition for 1 ITIMTCPO).
A similar effect has been reported by Dipple and Nebzy-doski (9) for TCPO concentrations of 40 to 120 /uM in asystem of mouse cells in culture. These authors believe thatunder these experimental conditions TCPO prevents formation of an intermediate metabolite, such as a trans-dihydrodiol, for example, without which the ultimate metab-
Table 2[3H]DBF binding to several DNA and tfÃNAsamples of diverse
originIn these determinations 0.3-mg/assay amounts of each nucleic
acid was used. Incubation lasted 40 min. The assay conditionswere the same as indicated in "Materials and Methods."
olite responsible for DNA binding cannot be produced.On the other hand, Selkirk ef al. (36) have shown that
TCPO does indeed inhibit dihydrodiol formation but alsoreduces BP metabolism down to 50% and significantlymodifies the metabolite proportions. TCPO could thereforealso inhibit monooxygenase activity and produce a reducing effect on [3H]DBF binding.
Similarly, CHO increased [3H]DBF binding to DNA in the
presence of rat liver but reduced this binding in the case ofmouse liver.
Despite well-known differences in AHH and epoxide hy-dratase content between rat and mouse liver, we presentlyhave insufficient data to interpret our observations withCHO.
The results obtained with DNA clearly showed a parallelbetween DBF binding and the GC pair content of thenucleic acids. With polyadenylate, polyinosinate, and poly-cytidylate no binding occurred, but with poly(G) efficientbinding of [3H]DBF was observed. Thus, guanine may beconsidered as the main site for DBF binding to the differentnucleic acids tested.
Analysis of the number of DBF molecules bound to thedifferent polyribonucleotide chains gives a proportion of 1molecule of DBF for 70,000 nucleotides in salmon spermDNA and for 22,600 nucleotides in poly(G). These valuesagree fairly well with those reported in the literature for BP,i.e., 1 BP per 50,000 nucleotides (11, 17, 43). That noselective losses occurred during DNA purification can bereasonably assumed from the results obtained with varioussynthetic polynucleotides.
Since [3H]DBF binding to DNA and poly(G) was resistant
to treatment by heat, alkali, or strong acids, one mayassume that it is covalent.
The question arises as to what kind of DBF metabolitesbind to DNA. DBF has no apparent K-region. Analysis ofDBF oxidation with osmic acid enabled Jacquignon ef al.(18) to show that this reaction takes place at the 5-5adouble bond, which possesses the strongest calculatedindex in the molecule. This oxidation progresses extremelyslowly. We may therefore consider the 5-5a bond as asterically hindered pseudo-K-region, which would explainits low reactivity (at least 10 times weaker than a true K-region).
None of the DBF metabolites isolated migrated chroma-tographically with model molecules corresponding topseudo-K-region diols.5 It is therefore reasonable to con
clude that the DBF metabolites bound to DNA are notepoxides of the pseudo-K-region.
Thus the most probable reacting metabolites might bethe epoxides or vicinal dihydrodiol-epoxides located on theA and/or D rings. This hypothesis agrees with current ideasabout the nature of ultimate carcinogenic PAH metabolites,such as those of BP where, according to several groups ofworkers, a non-K-region vicinal dihydrodiol-epoxide seemsto be responsible for in vivo binding of this PAH to cell DNA(3, 8,13, 15, 20, 31, 38). The bay-region theory of polycyclichydrocarbon carcinogenesis of Jerina ef al. (19) would alsopredict that the ultimate carcinogen of DBF might be the3,4-dihydrodiol-1,2-epoxide.
This interpretation should be taken with caution. In fact,under similar conditions, Thompson ef al. (41) showed that
important differences exist in the chemical nature of metabolites of 7-methylbenzanthracene bound to DMA whetherthis PAH is incubated with liver microsomes or in a systemcloser to in vivo conditions such as cultured mouse fibro-blasts. These differences probably originate in the very lowor nonexistent production of vicinal dihydrodiol-epoxidesin a 1-step/n vitro incubation.
In order to identify the nature of the DBF metabolitebound to DNA, we are currently analyzing enzymaticallyhydrolyzed DNA.
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)fluoranthene, a Carcinogenic, Polycyclica,eBinding of Dibenzo(
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