Chem.-Biol Interactions, 75 (1990) 131-140 Elsevier ScientificPublishers Ireland Ltd.

131

SEQUENCE S P E C I F I C I T Y IN T H E R E A C T I O N OF BENZOPYRENE DIOL E P O X I D E W I T H DNA

M A R T I N R. O S B O R N E

Institute of Cancer Research, Chester Beatty Laboratory, ~37 Fulham Road, London SW3 6JB IU.K.)

(Received November 7th, 1989) (Revision received February 6th, 1990) (Accepted February 14th, 1990) SUMMARY Benzopyrene diol epoxide (BPDE; (+)-7R,8S-dihydroxy-9S,lOR-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene), the ultimate carcinogen derived from the polycyclic hydrocarbon benzo[a]pyrene, reacts principally with the guanine bases in DNA. Nineteen double stranded, self-complementary oligonucleotides, containing deoxyguanosine in various sequence contexts, were each treated with tritium hbelled BPDE. The extent of reaction was determined by releasing the BPDE-guanine adduct with acid, isolating it by chromatography on a reverse-phase column, and estimating it by its radioactivity. Oligonucleotides containing an isolated guanine, such as AAGTACTT, were little affected by BPDE. Reactivity was increased where the guanine was flanked by another guanine on the same strand (e.g. TACCTAGGTA) or on the complementary strand (e.g. TATTCGAATA), and was highest in mixed G-C sequences such as ATCCGGAT. The results should help predict major sites of attack of BPDE on cellular proto-oncogenes.

K e y words: Benzo[a]pyrene -- Diol-epoxide -- Oligonucleotide -

Specificity

INTRODUCTION It has been suggested that in many instances, one step in the transformation of a mammalian cell into a malignant cell is the mutation of the ras proto-oncogene into an oncogene. Mutations in any one of the codons 12, 13, 59, 61 or 116 have been shown to be effective in such malignant transformation [1]. In accordance ~with this, malignant cell lines derived from many human tumours have been found to contain ras genes carrying mutations in codons 12, 13 or 61 [2]. Polycyclic hydrocarbons are effective in causing cell transformation in vitro, and cancer in rats and mice. It was therefore 0009-2797/90/$03.50

© 1990 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland

132 to be expected t h a t activated ras genes would be found in hydrocarbont r a n s f o r m e d cells. The m u t a t i o n s which have so far been o b s e r v e d are listed in Table I. The results are s o m e w h a t surprising, in t w o respects. Firstly, t h a t hydrocarbons such as d i m e t h y l b e n z a n t h r a c e n e and 3-methylcholanthrene, which are chemically similar, should cause m u t a t i o n s in different r a s genes. Secondly, in the limited n u m b e r of a c t i v a t i n g base changes found so far. The h y d r o c a r b o n s are k n o w n to cause m u t a t i o n s t h r o u g h their diol epoxide metabolites, and t h e s e r e a c t with guanine and adenine bases in the DNA. B e n z ~ p y r e n e diol epoxide r e a c t s principally with guanine [ 1 7 - 1 9 ] , while d i m e t h y l b e n z a n t h r a c e n e diol epoxide binds to both purines to a comparable e x t e n t [20]. One would t h e r e f o r e have e x p e c t e d to see a high e x t e n t of mutation in codons 12 and 13 (both GGN), r a t h e r than the bias t o w a r d s codon 61 which has been observed. T h e r e are several possible explanations for this selectivity, as outlined by Topal [1]. One is t h a t the carcinogen, in the form of its diol epoxide metabolite, shows sequence specificity in its reaction with the ras gene, attacking Ha-ras codon 61 (say) to a particularly high extent. A n o t h e r is t h a t modifications in some p a r t s of the gene escape the normal D N A repair process, because of some unusual s e c o n d a r y s t r u c t u r e at t h a t point.

TABLE I HYDROCARBON-INDUCEDMUTATION IN THE R A S GENES OF TRANSFORMED MAMMALIAN CELLS Column 4 shows the number of independently transformed cell lines having the indicated mutation, compared with the total number of such cell lines examined. Hydrocarbon

Tissue

Mutations

Frequency

Ref.

DMBA DMBA DMBA DMBA DMBA DMBA DMBA DMBA DBA BP BP 3MC 3MC 3MC 3MC 3MC 3MC

Mouse skin Mouse skin Mouse skin Mouse skin Mouse skin cells Mouse liver Mouse bladder Rat mammary Mouse skin Guinea pig cells Mouse lung Mouse cells Guinea pig cells Mouse thymomas Mouse sarcomas Mouse sarcomas Human cell line

Hra8 61 CAA to CTA Hras 61 CAA to CTA Hras 61 CAA to CTA Hras 61 CAA to CTA Hras 61 CAA to CTA Hras 61 CAA to CTA Kras 12 GGT to AGT Hra8 61 CAA to CTA Hra8 61 CAA to CTA Nras 61 CAA to CAT Kras 12 GGT to TGT Kras 12 GGT to TGT Nras 61 CAA to CAT Kras

3/3 33/37 15/21 2/2 11/20 4/5 2/23 5/6 1/1 8/16 4/5 1/1 10/12 4/6 2/6 1/1

3 4 5 6 7 8 9 10 3 11 12 13 11 14 15 15 16

Kras Nras

H~s 61

133

Alternatively, the differences may be due entirely to biological factors; for instance, one particular mutation may be particularly efficacious in producing the observed tumour in the tissue, species and strain of animal used. Here we consider the first possibility; namely, that the diol epoxide does not attack all the bases of one kind equally but shows sequence specificity. This paper reports the results of experiments with benzo[a]pyrene diol epoxide, whose reaction with DNA has been well characterised. We have examined the extent of reaction with double stranded oligonucleotides, containing the guanine base in various sequence contexts. For instance, the reactivity of benzopyrene diol epoxide with the octanucleotide d(AAGATCTT).d(AAGATCTT) gives information about the reactivity of guanine in the sequence context -AGA-. MATERIALS AND METHODS

Tritiated benzopyrene diol epoxide ((+)-anti isomer: 7R,8S-dihydroxy-9S, 10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene} was supplied by Mid-West Research Institute (Kansas City, MO) under contract from the Chemical Research Resources Branch, NCI (Bethesda, MD). A series of oligodeoxyribonucleotides, listed in Table II, were synthesized using an Applied Biosystems 381A DNA Synthesizer by the phosphoramidite method. They were purified by anion exchange chromatography (HPLC on a Whatman SAX column or FPLC on a Pharmacia Mono Q column).

Reaction of benzopyrene diol epoxide with oligonucleotides The oligonucleotide (0.50D units, approx. 15 ~g) in 0.5 ml sodium cacodylate buffer (0.02 M, pH 6.0) was warmed to 60 °C, then allowed to cool slowly to 0°C to maximise duplex formation. Tritiated epoxide (70 rig, 0.2 pCi) in 10 /~l dry dimethylformamide was added, and the mixture left overnight at 0 °C. 0.5 ml water was added, and the mixture was extracted ten times with 1 ml ether, which removed 99% of the hydrolysis products of the epoxide. The aqueous phase was acidified with hydrochloric acid (to 0.1 M HC1) and heated to 100°C for 10 rain. This released both native and modified guanine and adenine from the DNA; a small proportion of adducts may be destroyed by this treatment [17] but this should not affect the conclusions. The mixture was neutralized with sodium hydroxide and phosphate buffer, and analysed by reverse phase liquid chromatography, using one of the following systems: (i) A Pharmacia FPLC Pep-RPC column (50 x 5 ram) eluted with 43--46% methanol in water containing 0.005 M phosphate buffer (pH 7), 30 min at 0.85 ml/min. (ii) A Waters Microbondapak C18 column (250 x 4.6 ram), using a linear gradient of 3 5 - 5 5 % methanol in water, 30 rain, 2 ml/min. Fractions (1 or 2 rain) were collected and assayed for tritium radioactivity by liquid scintillation counting. RESULTS

Typical results are s h o w n in Fig. 1. T h e experiment in Fig. 1 A was a con-

134 1000-

A

500-

"~ ¢-

0

b

I

Q

E E

I

b

I

I

B

500.~_ .9

E

0

I

!

I

Q

2~

C 500 -

d

0 0

10

, 20

, 30

/+0

Etution time, minutes Fig. 1. Elution of [SH]-benzopyrene-diol-epoxide adducts from a Pep-RPC column, as determined by tritium activity (cpm; efficiency approx. 30°). The adducts were derived from the oligonucleotides (A) TATTATAATA; (B) TATGATCATA; (C) TATTCGAATA. The area under the graph gave the amount of tritium activity in each peak. Peak d corresponded to the principal epoxide-guanine adduct.

trol experiment, in which an oligonucleotide containing no guanine, d(TATTATAATA), was used. Similar results were obtained in other control experiments: with octathymidylic acid, d(TTTTTTTT), with the oligonucleotide omitted from the reaction mixture, and when the acid treatment was omitted. Peaks a and f appeared to be unrelated to reaction between benzopyrene diol epoxide and the oligonucleotide. Peak a contained polar material, including tritiated water, and peak f non-polar material eluted by methanol at the end of the run. Both arose, in part, from impurities in the tritiated

135 epoxide preparation. Peak b was benzopyrene-7,8,9,10-tetrahydrotetrol, i d e n t i f i e d b y t h e c o i n c i d e n c e of its e l u t i o n t i m e w i t h t h a t of a n a u t h e n t i c s a m p l e of u n l a b e l l e d t e t r o l . I t a r o s e f r o m h y d r o l y s i s of t h e diol e p o x i d e duri n g t h e r e a c t i o n ; s o m e 1 % of t h e t o t a l a c t i v i t y s u r v i v e d t h e t e n e t h e r e x t r a c t i o n s to g i v e a p e a k h e r e . P e a k e w a s a B P D E - a d e n i n e a d d u c t , as s h o w n b y t h e s i m i l a r i t y of its r e t e n t i o n t i m e t o t h a t of o n e of t h e p r o d u c t s of r e a c t i o n of b e n z o p y r e n e diol e p o x i d e w i t h [14C]-adenine, p r o b a b l y N 6 - B P D E - a d e n i n e . Figures 1B a n d 1C s h o w results with two guanine-containing o l i g o n u c l e o t i d e s . T h e e x t r a p e a k , d, c o r r e s p o n d e d to a B P D E - g u a n i n e a d d u c t , w h i c h has b e e n s h o w n t o b e N 2 - B P D E - g u a n i n e [17--19]. T h a t t h i s m a t e r i a l c o n t a i n e d g u a n i n e , a n d w a s n o t j u s t a n o t h e r h y d r o l y s i s p r o d u c t of t h e e p o x i d e , w a s s h o w n b y t h e f o l l o w i n g o b s e r v a t i o n s : (i) p r o t o n a t i o n in acid, as s h o w n b y l o w e r e x t r a c t a b i l i t y f r o m a q u e o u s s o l u t i o n a t p H < 3 [17] a n d (ii) f u r t h e r acid t r e a t m e n t c o n v e r t e d it t o B P - t e t r a h y d r o t e t r o l [17]. P e a k c w a s TABLE II EXTENT OF REACTION OF BENZOPYRENE DIOL EPOXIDE WITH SELF-COMPLEMENTARY OLIGONUCLEOTIDES Oligonucleotide•

Extent of reactionb

÷ G contentc

-~ 1. AACATGTT 2. TATGATCATA 3. AAGTACTT 4. ATAGTACTAT 5. AAGATCTT 6. AAACGTTT 7. TTTCGAAA 8. TATTCGAATA 9. TTTGCAAA 10. TATTGCAATA 11. AAAGCTTT 12. TACCTAGGTA 13. TGGATCCA 14. AAGGCCTT 15. ATGGCCAT 16. ATCCGGAT 17. TCCCATGGGA 18. AGGGTACCCT 19. TCGCATGCGA 20. Salmon sperm DNA

1.75 4.0 6.4 3.6 5.7 5.3 7.9 7" 9.2 9e 12.3 5.1 13.5 10.3 14.1 12.8 21.2 13.8 19.0 16.4 9.0

-

± ± ± ± ± ± ±

0.2 1.3 1.8 1.0 1.9 1.4 1.9

± 2.2 ± 3.7 ± 1.2 ± 2.0 ± 2.5 ± 4.1 _+ 3.1 ± 7.6 ± 3.3 ± 5 ± 2.9 ± 3.4

2.2 4.6 1.8 3.9 3.5 6.1 5 7.4 7 10.5 3.3 5.8 4.2 6.2 5.0 9.7 4.0 5.3 4.9 -

'Sequences are written from 5' to 3'. The oligonucleotides were self-complementary and would be expected to form dimers such as AACATGTT • TTGTACAA. bUnits: dpm of adduct (peak d) recovered per 1000 dpm diol epoxide used. 1 unit coresponded to about 1 adduct per 20 000 mol oligonucleotide. °Extent of reaction less control (1.8), divided by the number of guanines on one DNA strand. JSix control experiments, using no oligonucleotide, or oligonucleotides containing no guanines. "Single experiment, since only a small quantity of oligonucleotide was available. The other figures are average results from four or more experiments.

136

frequently seen, though often not clearly separated from b. It was probably a hydrolysis product of the diol epoxide. The extent of reaction, as determined from the size of peak d, is given in Table II. The blank value of 1.8 should be subtracted from all the other results to give the level of binding. The first 5 oligonucleotides, which have single guanines flanked by adenine or thymine, bound only 0.2-0.4o/o of the total epoxide added. The following 6 oligonucleotides (nos. 6 - 1 1 ) have a single guanine residue with a Cytosine next to it (or, perhaps more significantly, an adjacent guanine in the complementary strand). Here the level of reaction was higher, about 0.5--1°/o of the epoxide. The third group consists of 7 oligonucleotides (nos. 12--18) with 2 or 3 consecutive guanines, which bound 1 -2O/o of the added epoxide. This was generally greater than would be expected for 2 or 3 isolated guanine residues. Natural DNA bound the epoxide to an intermediate level, as would be expected for a mixture of the preceding sequences. DISCUSSION Benzopyrene and its diol epoxide do not bind to DNA perfectly randomly. This has been established by fractionating chromatin containing bound benzopyrene by partial nuclease digestion or by other methods, and estimating the benzopyrene in each fraction (summary of results in Ref. 21). It is not clear, however, how this apparent selectivity relates to the sequence of DNA, and whether it is of any consequence in determining the potency of benzopyrene in initiating tumours. The ready availability of synthetic oligonucleotides now gives an opportunity to try to determine sequence specificity rules, while knowledge of the base sequence of some proto-oncogenes may lead to the discovery of 'hot spots' which are especially prone to benzopyrene-induced mutations, which lead to cell transformation. Some carcinogens bind to DNA at the ring nitrogen atoms of the purine bases (principally 7-guanine). It is then relatively easy to determine the distribution of adducts on a fragment of a gene, by cleaving the DNA at the site of these adducts with piperidine, and then locating the cleavage points on a sequencing gel. This method has been successfully used to investigate sequence selectivity in the binding of nitrogen mustards and of aflatoxin B 1. The same method has been applied to the reaction of benzopyrene diol epoxide with DNA [22,23], leading to some conclusions concerning sequence selectivity; Lobanenkov [23] found that 83 of his 92 cleavage points corresponded to guanines whose 5'-neighbour was cytosine or thymine. But these conclusions relate only to attack of the epoxide at 7-guanine, and are unlikely to be relevant to mutation and cell transformation by benzopyrene, where it is probable that the biologically significant reactions are those involving the exocyclic amino groups of guanine and adenine [24,25]. To determine the distribution of stable adducts on a DNA sequence, indirect methods have to be used. The first of these was by a study of the mutations caused by these adducts. Eisenstadt [26] found four hot spots for

137 induction of amber or ochre mutations in the bzcI gene of E. coli by benzopyrene diol epoxide; these were caused by substitution of another base in place of guanine in the sequence contexts GCGAC, TCGTA, ATGAA or GTGAG. Vousden [27] found that of 13 mutants induced by benzopyrene diol epoxide in the Ha-ras gene, 2 had changes in codon 12 (GGC) and 11 had changes in codon 61 (CAG mutated to TAG (4), CTG (3), CAT (2), CCG or CAC). Yang [28] sequenced 93 benzopyrene diol epoxide induced mutations in a tRNA gene, and found two hot spots for mutation, both at the second guanine in GGGA sequences. The specificity in these instances may reflect sequence specificity in the reaction of the epoxide with DNA, but it is more likely that the specificity comes at a later stage -- in selective repair or misrepair of modified guanines, or because the efficiency with which an adduct causes mutations depends upon the sequence context. The most obvious chemical method for investigating sequence specificity in the reaction of benzopyrene diol epoxide with DNA is that employed here; to determine which of a set of short DNA sequences reacts with the epoxide to the greatest extent. The difficulty is to estimate the level of reaction unambiguously. It is not sufficient to isolate the oligonucleotide and measure bound hydrocarbon by its radioactivity or fluorescence; as shown in Fig. 1, much of the apparently bound material is not covalently bound. The DNA must be degraded and the product isolated. We have avoided enzymic hydrolysis, as we could not be sure that the enzymes would not themselves show sequence selectivity. The method used here, release of the products by acid hydrolysis, should be indiscriminate with regard to DNA sequence. The conditions have to be narrowly defined, however, as prolonged acid treatment would destroy the adducts. The results showed that binding of benzopyrene diol epoxide to guanine residues in DNA is enhanced by an adjacent cytosine or guanine. This enhancement was seen whichever side the cytosine was on (CG or GC). The highest level of binding was found with d(ATCCGGAT), which has a guanine base flanked by both cytosine and guanine. Binding was also high in oligonucleotides having triple guanines, though in those studied so far (nos. 17--19) not significantly higher than in those having double guanines. These conclusions are consistent with those of Chen [29] who found that the level of binding of benzopyrene diol epoxide to synthetic DNA decreased in the order poly dG-poly dC > poly(dG-dC)" poly(dC-dG) ~> poly(dG-dT)" poly(dC-dA). The data of Table II may be used to estimate to what extent various sites in protooncogenes may be susceptible to attack by BPDE. For example, likely sites of mutation of the mouse or rat c-Hras gene are codon 12, (T)GGA(G) and codon 61, (T)CAA(G). The guanines of codon 12 have a similar environment to those of our oligonucleotide no. 13, TGGATCCA; codon 61 has a guanine in the other strand, (C)TTG(A), similar to that in no. 2, TATGATCATA. These were sites of medium reactivity, no. 13 being more reactive than no. 2. This, then, should be the relative reactivity of codons 12 and 61 in c-Hras. The differences between different oligonucleotides in their extent of reaction were not great, but these differences may be magnified in

138 comparing two different reaction sites in the same molecule; a large difference in reaction rate between two sites may make only a small difference in the extent of reaction when carried through to completion, as was done here. These results may be compared with those of Reardon et al. [30] who studied the reaction of benzopyrene diol epoxide with fragments of the rat cHras proto-oncogene. Their method was to use the modified DNA as a template for DNA polymerase, and estimate the extent of reaction by the propensity of the polymerase to stop at that point. They found that the guanines of codon 12 (GGA) were modified to some extent but reaction at the codon 61 (other strand, TTG) was negligible. Other sites were attacked to a greater extent, e.g. the TGGGA sequence in codons 14--15 (compare oligonucleotide no. 17}. The method gives extensive data on DNA reaction specificity in a gene containing many varied guanine sites, but is open to the objection that the enzyme step may introduce sequence selectivity effects of its own. We have so far only considered reaction and mutation at translated sites, leading to an altered ras protein. The extent of reaction would be expected to be greater in regions of DNA containing longer tracts of consecutive GC or CG base pairs. Such tracts are found in an untranslated region to the 5' end of the gene, and this region has been found to be a site of attack by BPDE [31]. The region has ten GGCGGG 'boxes' which play some part in promoting the gene's activity [32], and these may be an important target of attack, as with the aprt gene [33]. ACKNOWLEDGEMENTS

Thanks to Prof. P. Brookes for his help and encouragement. The work was supported by grants to the Institute of Cancer Research from the Medical Research Council and the Cancer Research Campaign. REFERENCES 1 2 3

4 5

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