HPLC separation of 32P-postlabelled benzo[b]fluoranthene-DNA adducts

159

Cancer Letters, 65 (1992) 159- 167 Elsevier Scientific Publishers Ireland Ltd.

HPLC separation adducts W. Pfau‘

of 32P-postlabelled

, N.C. Hughes,

benzo[b]fluoranthene-DNA

P.L. Grover and D.H. Phillips

Haddow Laboratories, The Institute of Cancer Research, Cofswold Road, (Received

9 March

(Accepted

2 June 1992)

Surrey

SM2

5lVG (UK)

1992)

Summary

pathway for BbF activation in skin probably involves a bayregion triol-epoxide possessing a phenolic OHgroup on the peninsula ring.

dicate that the predominant

Analysis using 32P-postlabelling recently deueloped HPLC method the adduct formed by reaction

and

a

resolved

of the anti-bay-region the more polar major adduct produced by the hydrocarbon in three different biological systems. In each case, the adduct formed from the anti-bay-region dial-epoxide constituted only a minor proportion of the total DNA modification. Comparisons of the DNA adducts formed from the hydrocarbon with those formed in microsomal incubations from the putative metabolites BbF-9,10-dial, anti-BbF-9,lO-dial-11,12-0xide and the 5,9,10- and 6,9,10-BbF-trials inbenzo[blfluoranthene (BbF) dial-epoxide with DNA from

Correspondence to: D.H. Phillips, Haddow Laboratories, The Institute of Cancer Research, Cotswold Road, Sutton, Surrey SM2 5NG, UK. Abbreofottons: SS-DNA, salmon sperm DNA; PAH, poiycyclic aromatic hydrocarbon; 2-OH-BbF, P-hydroxybenzo[b]fluoranthene; 3-OH-BbF, 3-hydroxybenzo[b]fluoranthene; 6-OHWF, 6-hydroxybenzo[b]fluoranthene; 7-OH-BbF, ir-hydroxybenzo[b]fluoranthene; BbF-9, lo-diol, trans-9, IO-dihydro-9, lodihydroxybenzo[b]fluoranthene; BbF-l,P-dial, trans-1,2dihydro-1,2-dihydroxybenzo[b]fluoranthene; anti-BbF-9, lodial-11,12-oxide, r-9,t-lo-dihydroxyt-11,12-oxy-9,10,11,12tetrahydrobenzo[b]fluoranthene; BbF-5,9,10-triol, 5-hydroxytrans-9,lO-dihydro-9,lO-dihydroxybenzo[b]fluoranthene; BbF6,9,10-triol, 6-hydroxy-trans-9, IO-dihydro-9, lo-dihydroxybenzo[b]fluoranthene. ‘Present address: Department of Toxicology, University of Hamburg Medical School, Grindelallee 117, 2000 Hamburg 13, Germany.

0304-3835/92/$05.00 Printed and Published

Sutton,

0 1992 Elsevier Scientific Publishers in Ireland

Keywords: benzo[b]fluoranthene; activation; DNA adducts; skin

metabolic

Introduction Benzo[b]fluoranthene (BbF) , a non-alternant PAH, is a ubiquitous environmental pollutant [l] and a suspected human carcinogen [Z]. As a tumour initiator in mouse skin it is less potent than benzo[a]pyrene but more active than chrysene or dibenz[a,h]anthracene [2,3]. The molecular structure features a bay-region between Cl and Cl2 in the phenanthrene part of the molecule and a so-called peninsula ring (C4 - C7). Amin, Hecht and coworkers synthesized a number of phenolic and dihydrodiol derivatives of BbF as potential metabolites [3-51 and identified the major metabolites formed by rat liver homogenate as being similar to those formed in vivo in the epidermis of topically-treated mice [6,7]. Surprisingly, no evidence was found for the formation of the bay-region diolepoxide , r-9,t-lo-dihydroxy-t-11,12-oxy9,10,11,12tetrahydrobenzo[b]fluoranthene (anti-BbF-9, IO-diol-11,12-oxide), its hydrolysis product, the 9,10,11,12-tetrahydrotetrol, Ireland

Ltd.

160

or its precursor, trans-9,10-dihydroxy-9, lodihydrobenzo[b]fluoranthene (BbF-9, lo-diol) . In comparisons between the tumourigenicity of the parent PAH, the major observed metabolites, the synthetic anti-BbF-9, lo-diol11,12-oxide, BbF-9, lo-diol and its metabolites (a mixture of 5,9, lo- and 6,9, lo-triols), only BbF-9, lo-diol showed higher tumourinitiating activity on mouse skin than BbF itself 171. 32P-Postlabelling is currently the most convenient method for the analysis of the low levels of DNA adducts and their analysis of DNA adducts can reveal information about the pathways of metabolic activation [8]. This assay has been employed before to analyse the DNA adducts formed from BbF in mouse skin [9] and by the bay-region diol-epoxide in vitro [5]. Here we report on the comparison of DNA adducts formed from BbF in the skin of topically-treated mice and in cultured human skin and by BbF, BbF-9,10-diol, the 5,9,10and 6,9, lo-triols and anti-BbF-9, lo-diol11,12-oxide in vitro in the presence of rat liver microsomes. A newly developed HPLC system has been used to analyse 32P-labelled BbF-nucleoside bisphosphate adducts [lo] with significantly improved resolution compared to the thin-layer chromatography procedure [8]. This new method allowed us to distinguish between the adducts formed by the different derivatives of BbF. Materials and Methods Instiumentation The HPLC apparatus consisted of two Waters 501 HPLC pumps, a Waters 712 WISP autosampler, a Waters 440 absorbance detector at 280 nm and a Berthold LB 507 A HPLC radioactivity monitor. Gradient control and data processing were achieved with a Waters Datastation with Wate’rs Baseline 810 software. Separations were performed on Zorbax phenyl-modified reversed phase columns (particle size 5 pm, 250 x 4.6 mm I.D.) supplied by Hichrom Ltd, Reading, UK. Fluorescence spectra were recorded on a

Perkin-Elmer MPF3L spectrofluorimeter. The mass spectrometer used was ? TSQ 700 triple quadrupole system (Finnegan MAT, San Jose) equipped with an electrospray ion source (Analytica, CT., USA). Chemicals and enzymes BbF was supplied by the Community Bureau of Reference (BCR, Brussels, Belgium). 2-Hydroxybenzo[b]fluoranthene (2OH-BbF), 3-hydroxybenzo[b]fluoranthene (3OH-BbF), 6-hydroxybenzo[b]fluoranthene (6OH-BbF), 7-hydroxybenzo[b]fluoranthene (7OH-BbF), trans-9,10-dihydro-9, lo-dihydroxybenzo[b]fluoranthene (BbF-9,10-dial), trans1,2-dihydro-1,2-dihydroxybenzo[b]fluoranthene (BbF-1,2-diol) and r-9,t-lo-dihydroxy-t11,12-oxy-9,10,11,12tetrahydrobenzo[b]fluoranthene (anti-BbF-9,10-diol-11,12-0xide) were obtained from the NC1 Chemical Carcinogen Standard Repository, NIH, Bethesda, MD, USA. Microsomes were obtained from livers of male Sprague - Dawley rats pretreated with 3-methylcholanthrene, and S9-homogenate was prepared from livers of male Sprague - Dawley rats pretreated with Arochlor according to Schneider and Hogeboom [ll], as modified by Lecoq et al. [12]. All other chemi.cals and enzymes were purchased from commercial sources. In vitro reactions

Anti-BbF-9,10-diol-11,12-oxide was dissolved by sonication in anhydrous ethanol and an aliquot of 140 nmol in 200 ~1 added to a solution of salmon sperm DNA (SS-DNA) (2 mg = 3 pmol) in 0.01 M Tris buffer, pH 7.4 (2 ml). The mixture was incubated overnight at room temperature, then extracted six times with diethyl ether and the DNA precipitated with NaCl and cold ethanol. Microsomal incubations were performed according to a protocol published before [12]; briefly, 1 pmol of BbF or a metabolite in 50 ~1 DMSO was added to a mixture of 4 mg NADPH, 5 mg MgClz and 6 mg microsomal protein and 1 mg SS-DNA in 2 ml Tris buffer (10 mM, pH 7.4). Incubation at 37OC was for

161

45 min in a shaking water bath. The reaction was stopped by extracting the mixture with 5 ml cold ethyl acetate and the DNA was reisolated as described [ 131. Preparation of the 5/6-hydroxy-9,lOdihydrodiol of BbF BbF-9,10-dial was incubated with rat liver homogenate (S9) from Arochlor-pretreated rats and cofactors as described above and as according to Geddie et al. [7]. The ethyl acetate extraction yielded as a major component a mixture of BbF-5,9,10-trio1 and BbF6,9,10-triol. This product was isolated by HPLC using an octadecyl-modified reversed phase column with a gradient of methanol in water [7]. The UV absorbance and fluorescence spectra (in ethanolic solution) showed maxima at 384 nm, 314 nm and 247 nm with shoulders at 410 nm, 365 nm, 355 nm and 304 nm; the shoulder at 304 nm shifted to a peak at 326 nm with the addition of alkali. Electrospray ionisation mass spectral analysis featured signals at 325 m/e (M + Na+) and 285 (M - Hz0 + H +), consistent with the compound being a phenolic derivative of a BbF dihydrodiol. Topical treatment of mice in uiuo The backs of Parkes mice (6 - 8 weeks old) were shaved and groups of four animals were treated with BbF (1 pmol) applied topically in acetone (200 ~1)to the shaved areas of skin. A control group was treated with acetone alone. Twenty-four hours later, the animals were killed by cervical dislocation, the treated areas of dorsal skin frozen in liquid nitrogen and the dermal surface scraped to remove excess fat. The frozen skin was powdered by sequential use of Atomix (MSE Ltd, Crawley, Sussex, UK) and Ultraturrax blenders (Sartorius Instruments, Belmont, Surrey, UK). Treatment of human skin samples in culture Samples of normal adult human skin from two patients undergoing reduction mammoplasty were obtained through the Depart-

ment of Pathology, Royal Marsden Hospital. Excess subcutaneous fat and connective tissue were removed and skin samples were dissected into 12-cm2 sections. They were then placed in culture as described previously [14,15]. For each of the two original skin samples, one portion was treated with acetone and two other portions were treated with 1 pmol BbF. Following treatment, the skin was maintained at 37OC for 24 h and then stored at -2OOC until the DNA was isolated. 32P-Postlabelling analysis DNA was isolated by the phenol extraction method of Kirby [ 161 with modifications according to Gupta [ 171. 32P-Postlabelling was performed by the nuclease Pl-enhanced procedure [18]: 4 pg of modified DNA dissolved in 5 ~1 buffer containing sodium succinate (20 mM) and calcium chloride (8 mM) was digested with micrococcal nuclease (2 pg) and spleen phosphodiesterase (2 pg) to 3 ’ phosphonucleosides and then further digested with nuclease Pl [18]. Labelling was performed with [T-~~P]ATP (25 &i, 3500 Ci/mmol) and T4 polynucleotide kinase (2.5 units) in a buffer (10 ~1, pH 9.0) consisting of bicineNaOH (10 mM) , MgClz (10 mM) dithiothreitol (10 mM) and spermidine (10 mM). Samples were applied to PEI-cellulose sheets (Macherey and Nagel, Di.iren, Germany) and eluted overnight with 2.3 M NaH2P04 (pH 6.0). The origin (1 cm2) was cut out, extracted for 12 h with 400 ,ul4 M pyridinium formate (pH 4.0), the extract evaporated under reduced pressure and aliquots used for HPLC separations. Separations were performed on Zorbax phenyl-modified reversed-phase columns (particle size 5 pm, 250 x 4.6 mm I.D.) eluted with a gradient of methanol in sodium phosphate buffer (buffer A, 0.3 M sodium dihydrogen orthophosphate and 0.2 M orthophosphoric acid, adjusted to pH 2.0; buffer B, 90% methanol, 10% buffer A) as follows: a linear gradient from 15% to 33% B at 15 min; linear gradient to 46% B at 60 min and to 80% B at 80 min at a flow rate of 1 ml/min.

162

ferent activation systems were also carried out to confirm that adduct peaks with similar retention times did coelute. HPLC elution profiles obtained from these experiments are shown in Fig. 1. Incubation of BbF with microsomes prepared from livers of 3-methylcholanthrene-pretreated rats in the presence of NADPH and SS-DNA produced a number of DNA adduct peaks (Fig. la). The major peak I eluted at 32 min and accounted for 70% of the total radioactivity, and minor peaks were observed at 27 min, 29 min, 34 min and 62 min.

R~UltS DNA adducts formed from BbF in the skin of topically-treated mice, in human skin maintained in short-term organ culture and with SSDNA in vitro in the presence of rat liver microsomes have been compared, together with the DNA adducts formed by BbF in the in vitro microsomal system and with adducts obtained from incubations of derivatives and metabolites of BbF. Where appropriate, cochromatography studies of mixtures of adducts formed by different metabolites or in dif-

11

a

I

II

i.i;

20 Fig. 1.

40

60 mm

20

40

80

mln

20

40

bu

min

Reversed phase HPLC profiles of 32P-labe11ed digests of DNA modified with BbF (a) in vitro with rat liver microsomes, (b) in vivo in mouse skin, (c) in human skin maintained in short-term organ culture. Profiles of 32P-labelled adducts obtained from digests of DNA incubated in the presence of rat liver microsomes with: (d) BbF-9,10-dial, (e) -OH-BbF, (f) 7-OH-BbF, (g) 5/6-g, lo-triol-BbF and (h) anti-BbF-9, lo-diol- 11,lZoxide are also shown. (i) Shows the profile obtained from a 32P-1abelled digest of DNA modified directly with anti-BbF-9,10-diol-l l, IZoxide. HPLC elution conditions were as described in Materials and Methods. Radioactivity was measured as Cerenkov radiation with an on-line detector. These profiles have not been standardised and do not reflect adduct levels.

163

The topical application of BbF in vivo to the dorsal skin of mice resulted in the formation of a major adduct in the epidermal DNA, as shown in Fig. lb, that accounted for 68% of the radioactivity and had a retention time of 32 min. A minor adduct (24%) eluted at 62 min; these adducts cochromatographed with adduct peaks I and II, respectively, that were formed in microsomal incubations with BbF (Fig. la). Human skin maintained in short-term organ culture and treated with BbF yielded three adduct peaks (Fig. lc) that eluted at 32 min, 49 min and 63 min and that accounted for 58%) 28% and 6%, respectively, of the total binding. The major peak co-eluted with peak I (Fig. la), the peak at 63 min co-eluted with the major product of the direct reaction of DNA anti-BbF-9,10-diol-11,12-oxide and (see below and Fig. li, peak II). The peak at 49 min was tentatively assigned as corresponding to a minor product formed by direct modification of DNA with the anti-BbF-9, IO-diol-11,12-oxide (Fig. li). Incubation of BbF-9,10-dial with SS-DNA in the presence of rat liver microsomes led to the formation of several adduct peaks (Fig. Id). Three major adduct peaks were observed, numbered Ia eluting at 28 min, I (32 min) and II (62 min) and these accounted for 408, 30% and 20%, respectively, of the total binding. When incubated with microsomes and DNA, 3-OH-BbF and 7-OH-BbF formed small amounts of adducts (Figs. le and If) which did not co-elute with the major adducts observed with the parent hydrocarbon and no adduct formation was observed with BbF-1,2-diol, 2OH-BbF or 6-OH-BbF, or in control experiments with DMSO alone (data not shown) in the presence of a microsomal activation system. When the mixture of BbF-5,9,10-trio1 and BbF-6,9,10-trio1 was incubated with a microsomal metabolising system in the presence of DNA, only early-eluting adduct peaks were observed as shown in Fig. lg. Although present in different proportions, these had retention times identical with those

of the two adducts formed from BbF-9,10dial, peak Ia at 28 min and peak I at 32 min; but there was no evidence for the formation of the DNA adduct eluting at 62 min. The formation of two major early eluting adducts could reflect the fact that a mixture of triols with phenolic groups at position 5 or 6 was used, or it could be due to diastereomeric isomerism since a racemic mixture of the synthetic BbF9,10-dial was employed to produce the triols. Direct reaction of anti-BbF-9, lo-diol- 11,12oxide with SS-DNA (Fig. li) resulted in one major adduct, peak II, with a retention time of 62 min and a minor peak at 50 min (> l%), while inclusion of a microsomal activation system led to the additional formation of an adduct peak I eluting at 32 min (Fig. lh) . Discussion BbF is one of the most abundant carcinogenic PAH in the environment [2] but despite a considerable amount of work, the pathways involved in the metabolic activation of this compound have remained obscure [4- 7,19,20]. It has been predicted from the bay-region theory that the activation of BbF might proceed via the 9, lo-dihydrodiol11,12-epoxide, the bay-region dial-epoxide. However, studies on the metabolism of BbF have failed to confirm this hypothesis [7]. In the present work we have investigated the formation of DNA adducts from BbF and some potential metabolites using a recently developed HPLC version of the 32P-pastIabelling assay. Whilst adduct resolution could not, be achieved on ion-exchange thin-layer chromatography (data not shown), reversed phase HPLC allowed the major DNA adduct formed upon direct reaction of the synthetic dial-epoxide with DNA in vitro to be separated from the major adduct that was formed from the parent hydrocarbon, BbF, either in vitro upon microsomal activation or in vivo in the epidermis of mice treated topically with BbF. The chromatographic properties suggested that the latter adduct was more polar, probably

164

because of the presence of an additional hydroxyl group. The HPLC elution profiles obtained when BbF was activated to DNA-binding species in vivo in mouse epidermis or in vitro in the presence of rat liver microsomes were similar, with a major adduct peak I and a minor, Iatereluting peak II (Figs. la and lb). This tends to confirm the results published by Geddie et al. [7] which showed that the metabolism of BbF is essentially the same in vitro using rat liver homogenate and in vivo in mouse epidermis. However, in addition to these adducts, a third peak was observed in DNA from human skin samples treated in vitro with BbF (Fig. lc): this product has not been further examined but has a retention time similar to that of a minor adduct formed from anti-BbF-9, lo-diol- 11,12oxide (Fig. li). Peak II was the major adduct observed synthetic anti-BbF-9,10-diol-ll,lZwhen oxide reacted directly with DNA, therefore the hydrocarbon moiety in this adduct is presumed to have a r-9,t-lO,t-ll-trihydroxy-9,10,11,12tetrahydrobenzo[b]fluoranthene structure that is probably linked via Cl2 to the exocyclic amino group of deoxyguanosine or deoxyadenosine. Peak I and peak II were both observed in microsomal incubations of DNA with anti-BbF-9, lo-diol- 11,12-oxide, BbF9,10-diol and with the hydrocarbon itself whereas incubation of the mixture of triols gave only the more polar adducts Ia and I. The conclusion to be drawn from these data is that the earlier eluting peaks la and I have the hydrocarbon moiety in the form of a 5- or 6,9,10,11-tetrahydroxy-9,10,11,12-tetrahydrobenzo[b]fluoranthene moiety, i.e. a structure which, compared to adduct II, possesses an extra phenolic OH-group. These results indicate that at least two different ultimate carcinogens are probably responsible for the DNA binding of BbF that occurs in both mouse skin and microsomal incubations. Reaction of the bay-region diolepoxide gives rise to adduct II, which contributes, as a minor adduct, to the covalent modification of DNA by BbF in the three dif-

ferent biological systems investigated in this study. Secondly, in what appears to be the major pathway of activation, we postulate the intermediate existence of a 5- and/or 6hydroxy-9,10,11,12-tetrahydro-9, lodihydroxy- 11,12-oxy-BbF, whose reactions lead to the formation of the more polar DNA adduct I. The latter adduct accounts for two thirds of the total binding that occurs with BbF in vivo and in vitro. These two proposed pathways for the metabolic activation of BbF are summarized in Fig. 2. Geddie and co-workers [7] investigated the metabolism of BbF in vitro with microsomes from rat liver induced with Arochlor and in vivo in mouse epidermis using 3H-labeIled BbF. They observed the formation of phenolic metabolites, namely 4-, 5-, 6-, 7- and llhydroxy-BbF with hydroxylation on the peninsula ring being the major pathway of metabolism. By means of chromatographic comparisons with authentic standards, BbF1,2-diol and the 11,12-dihydrodiol were identified as the major dihydrodiol metabolites, but no evidence for the formation of the precursor dihydrodiol of the bay-region diol-epoxide, BbF-9,10-dial, or of the corresponding tetrol was obtained [7]. When synthetic BbF-9, lodiol was incubated in excess with rat liver microsomes, a new metabolic product, composed of a mixture of BbF-5,9,10-trio1 and BbF-6,9, lo-trio], was observed. However, these triols were not detected in vitro or in mouse epidermis when the metabolites of t3H]BbF were analysed. Problems in the detection of intermediate metabolites can be due to kinetic effects, i.e. the rate of formation is slower than the rate at which further metabolism can occur. This has been reported, for example, in the search for a similar trio], the 9-hydroxy- 1,2-dihydrodiol of chrysene and evidence for the formation of this metabolite was eventually obtained in trapping experiments using 3H-labelled chrysene and the unlabelled trio1 [21]. During the course of the present study, tritiated BbF was not available to us but similar experiments are planned in order to verify the proposed

165 2

-Ho.r-$?IQ Hor)&=J t t OH

OH

ADDUCT II

ADDUCT I

Fig. 2. Proposed metabolic activation pathways for BbF involving a bay-region dial-epoxide and a triol-epoxide as DNA binding species that lead to adducts II and I, respectively.

pathways of metabolic activation of BbF. Investigating the tumour initiating activity of several metabolites of BbF on mouse skin, Geddie et al. [7] observed an increased activity for the dihydrodiol compared to the BbF itself and this is in agreement with our DNA binding results; in contrast to our findings on DNA binding activity, however, neither the 5-/6hydroxy-9, lo-dihydrodiol nor the bay-region diol-epoxide seemed to be as carcinogenic as the parent compound [7]. As has been reported for a number of other PAHs the dihydrodiol is often the most active metabolite [22], whilst the corresponding diol-epoxides

may be too reactive, i.e. unstable, to reach their target. DNA binding profiles obtained in vivo in mouse skin and in vitro with rat liver microsomes were very similar. However, an important goal of studies on the metabolism of chemical carcinogens is to elucidate the pathways concerned with bioactivation in humans. For obvious ethical reasons only in vitro experiments are possible using human tissue. The results presented in this study suggest that, for the DNA binding of BbF, extrapolation from animal data to the human situation may be permissible to some extent.

166

In summary, these studies demonstrate that BbF is metabolically activated via a metabolite that is more polar than a simple diol-epoxide. Whilst the major adduct is chromatographically characteristic of one formed by a triol-epoxide, its definitive characterisation will require the provision of synthetic standards, the preparation of which is currently in progress. Acknowledgements

The authors thank Dr. S. Lecoq for the preparation of rat liver microsomes and S9homogenate and Dr. G.K. Poon for performing mass spectral analysis. This work was supported, in part, by grants from the Medical Research Council and the Cancer Research Campaign and, in part, by PHS grant no. CA 21959 from the US National Cancer Institute, DHHS.

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1579 - 1584. Phillips, D.H. (1989) Modern methods of DNA adduct determination. In: Chemical Carcinogenesis and Mutagenesis I, pp. 503-546. Editors: C.S. Cooper and P.L. Grover. Springer - Verlag, Heidelberg, Germany. 9 Weyand, E.H., Rice, J.E. and LaVoie, E.J. (1987) 32Ppostlabeling analysis of DNA adducts from non-alternant PAH using thin-layer and high performance liquid chromatography. Cancer Len., 37, 257 - 266. 10 Pfau, W. and Phillips, D.H. (1991) Improved reversedphase high-performance liquid chromatographic separation of 32P-labelled nucleoside 3’,5’-bisphosphate adducts of polycyclic aromatic hydrocarbons. J. Chromatogr., 570, 65 - 76. 11 Schneider, W.C. and Hogeboom, G.N. (1950) Intracellular distribution of enzymes. V. Further studies on the distribution of cytochrome c in rat liver homogenates. J. Biol. Chem., 183, 123- 128. 12 Lecoq, S., Perin, F., Plessis, M.J., Strapelias, H. and Duquesne, M. (1989) Comparisons of the in vitro metabolisms and mutagenicities of dibenz[a,c]anthracene, indibenz[o,h]anthracene and dibenz[oj]anthracene: fluence of norharman. Carcinogenesis, 10, 461- 469. 13 MacNicoll, A.D., Cooper, C.S., Ribeiro, O., Gervasi, P.G., Hewer, A., Walsh, C., Grover, P.L. and Sims, P. (1979) The involvement of a non-‘bay-region’ diol-epoxide in the formation of benz[a]anthracene-DNA adducts in a rat-liver microsomal system. Biochem. Biophys. Res. Commun., 91,490-497. 14 Trowel, O.A. (1954) A modified technique for organ cell culture in vitro. Exp. Cell Res., 6, 236-251. 15 Weston, A., Hodgson, R.M., Hewer, A.J., Kuroda, R. and Grover, P.L. (1985) Comparative studies of the metabolic activation of chrysene in rodent and human skin. Chem.-Biol. Interact., 54, 223 - 242. 16 Kirby, K.S. (1957) A new method for the isolation of deoxyribonucleic acid: evidence on the nature of bonds between deoxyribonucleic acid and protein. Biochem. J., 66, 495 - 504. 17 Gupta, R.C. (1984) Nonrandom binding of the carcinogen N-hydroxy-2-acetylaminofluorene to repetitive sequences of rat liver DNA in vivo. Proc. Natl. Acad. Sci. USA, 81, 6943 - 6947. 18 Reddy, M.V. and Randerath, K. (1986) Nuclease PImediated enhancement of sensitivity of 32P-postlabeling test for structurally diverse DNA adducts. Carcinogenesis, 7, 1543- 1551. 19 LaVoie, E.J., Amin, S., Hecht, S.S., Furuya, K. and Hoffmann, D. (1982) Tumour initiating activity of dihydrodiols of benzo[b]fluoranthene, benzo[i]fluoranthene and benzo[k]fluoranthene. Carcinogenesis, 3, 49-52. 20 Shimada, T., Martin, M.V., Pruess-Schwartz, D., Marnett, L.J. and Guengerich, F.P. (1989) Roles of individual 8

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human cytochrome P-450 enzymes in the bioacttvation of benzo[a]pyrene, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene and other dihydrodiol derivatives of polycyclic aromatic hydrocarbons. Cancer Res., 49, 6304- 6312. Hodgson, R.M., Weston, A., Seidel, A., Bochnitschek, W., Glatt, H.R., Oesch, F. and Grover, P.L. (1985) The formation of 9-hydroxychrysene-l,P-diol as intermediate

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in the metabolic activation of chrysene. Carcinogenesis, 6, 135 - 139. Hall, M. and Grover, P.L. (1990) Polycyclic aromatic hydrocarbons: metabolism, activation and tumour initiation. In: Chemical Carcinogenesis and Mutagenesis I., pp. 327-372. Editors: C.S. Cooper and P.L. Grover. Springer-Verlag. Heidelberg, Germany,