Metallothionein crypt-restricted immunopositivity indices (MTCRII) correlate with aberrant crypt foci (ACF) in mouse colon.

Metallothionein (MT) crypt-restricted immunopositivity indices (MTCRII) are colonic crypt stem cell mutation markers that may be induced early and in abundance after mutagen treatment. Metallothionein is the endogenous reporter gene for MTCRII, but is not typically implicated in the classical pathway of colorectal tumorigenesis. Hence, the oncological relevance of MTCRII is unclear. This study tests the hypothesis that MTCRII induced by N-methyl-N-nitrosourea (MNU) and lambda carrageenan (lambdaCgN) associate with aberrant crypt foci (ACF) in mouse colon. Undegraded lambdaCgN and MNU were tested alone and in combination against MTCRII and ACF in Balb/c mice, at 20 weeks after the start of treatment. MTCRII were unaffected by lambdaCgN alone. Combined lambdaCgN/MNU treatments induced greater MTCRII (P < 0.01) as well as greater number (P < 0.001) and crypt multiplicity (P < 0.01) of ACF than MNU alone. MTCRII were approximately 10-fold more numerous than ACF, although linear correlations were observed between these parameters (r = 0.732; P < 0.01). MTCRII are induced by lambdaCgN/MNU interactions in sufficient numbers to provide statistical power from relatively small sample sizes and correlate with ACF formation. MTCRII could thus provide the basis for a novel medium-term murine bioassay relevant to early-stage colorectal tumorigenesis.

Resolution of alpha-hydroxytamoxifen; R-isomer forms more DNA adducts in rat liver cells.

The genotoxic tamoxifen metabolite alpha-hydroxytamoxifen has been resolved into R- and S-enantiomers. This was achieved by preparing its ester with S-camphanic acid, chromatographic separation into two diastereoisomers, and hydrolysis to give (+)- and (-)-alpha-hydroxytamoxifen. The configuration of the (-)-isomer was shown to be S- by degradation of an ester to a derivative of (-)-2-hydroxy-1-phenyl-1-propanone, which has already been shown to have S-configuration. Metabolism of tamoxifen by rat liver microsomes gave equal amounts of the two enantiomers. They have the same chemical properties but, on treatment of rat hepatocytes in culture, R-(+)-alpha-hydroxytamoxifen gave at least eight times as many DNA adducts as the S-(-)-isomer.

Preparation of a methylated DNA standard, and its stability on storage.

Several methods are available for the determination of low levels of O(6)-methylguanine in DNA; this base arises after methylation by environmental carcinogens. The reliability of these assays is much improved by the use of a standard. We have prepared such a standard by treating calf thymus DNA with [(3)H-methyl]-N-methyl-N-nitrosourea. We characterized the methylated bases by hydrolysis of a sample to nucleosides, followed by liquid chromatography and liquid scintillation counting of the tritium content. The level of O(6)-methylguanine was 0.6 per one million nucleotides. This base is stable, and its level was unchanged after storage for 5 years at -20 degrees C. The methylated DNA also contained 7-methylguanine and 3-methyladenine, which are slowly cleaved from the DNA on standing. The half-life for loss of 7-methylguanine at neutral pH was estimated to be 70 h at 39 degrees C, 460 h at 22 degrees C, 3800 h at 10 degrees C, and about 4 years at -20 degrees C.

Tamoxifen-DNA adduct formation in rat liver determined by immunoassay and 32P-postlabeling.

Tamoxifen (TAM), a nonsteroidal antiestrogen used as a chemotherapeutic and chemopreventive agent for breast cancer, induces liver tumors in rodents and covalent DNA adduct formation in hepatic DNA. Here, we report the development and validation of highly sensitive and specific immunoassays for the determination of TAM-DNA adducts. Rabbits were immunized with calf thymus DNA, chemically modified with alpha-acetoxytamoxifen to 2.4 adducts per 100 nucleotides, and the resulting antisera were characterized by competitive dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) and chemiluminescence immunoassay (CIA). Compared with DELFIA, the CIA has a much lower background and a 20-fold increase in sensitivity. For the immunogen TAM-DNA, 50% inhibition was at 2.0 +/- 0.11 (mean +/- SE, n = 18) fmol of (E)-alpha-(N2-deoxyguanosinyl)tamoxifen (TAM-dG) adduct in TAM-DNA by DELFIA. For TAM-DNA modified to 4.8 adducts in 10(6) nucleotides, 50% inhibition was at 20.6 +/- 6.6 (mean +/- SE, n = 8) fmol of TAM-dG in TAM-DNA by DELFIA and at 0.92 +/- 0.11 (mean +/- SE, n = 10) fmol of TAM-dG in TAM-DNA by CIA. No inhibition was observed in either assay with up to 20 microg (62.5 nmol of nucleotides) of unmodified DNA. The individual adducts TAM-dG and (Z)-alpha-(N2-deoxyguanosinyl)tamoxifen and the individual compounds TAM and 4-OH-TAM gave DELFIA 50% inhibitions at 828, 2229, 5440, and 8250 fmol, respectively. For assay validation, TAM-dG levels were determined by DELFIA, CIA, and 32P-postlabeling in TAM-DNA samples modified in vitro to different levels, and comparable values were obtained in all three assays. Further validation was obtained in vivo in rat liver. DNA adducts of TAM were measurable in rat liver 24 h after a single i.p. dose of 45 mg TAM/kg body weight and after daily p.o. dosing for 7 days with 5.0, 10.0, and 20.0 mg TAM/kg body weight. In addition, TAM-DNA adducts disappeared slowly over 21 days in rats on a control diet that were first given p.o. TAM at 45 mg/kg/day for 4 days. In the rat experiments, TAM-DNA adduct levels determined by CIA compared well with those determined by 32P-postlabeling, although the CIA gave an underestimation at the highest doses. For rat liver samples, the detection limit by CIA was 3 adducts per 10(9) nucleotides (0.2 fmol of adducts per 20 microg of DNA).

N-demethylation accompanies alpha-hydroxylation in the metabolic activation of tamoxifen in rat liver cells.

Previous work has shown that a major route of activation of tamoxifen to DNA-binding products in rat liver cells is via alpha-hydroxylation leading to modification of the N(2)-position of guanine in DNA and to a lesser extent the N(6)-position of adenine. Improved resolution by HPLC has now identified two major adducts in rat liver DNA, one of them the aforementioned tamoxifen-N(2)-guanine adduct and the other the equivalent adduct in which the tamoxifen moiety has lost a methyl group. Treatment of rats or rat hepatocytes with N-desmethyltamoxifen gave rise to the second adduct, whereas treatment with tamoxifen or alpha-hydroxytamoxifen gave rise to both. Furthermore, N,N-didesmethyltamoxifen was found to be responsible for an additional minor DNA adduct formed by tamoxifen, alpha-hydroxytamoxifen and N-desmethyltamoxifen. The involvement of metabolism at the alpha position was confirmed in experiments in which [alpha-D(2)-ethyl]tamoxifen, but not [beta-D(3)-ethyl]tamoxifen, produced reduced levels of DNA adducts. Tamoxifen N-oxide and alpha-hydroxytamoxifen N-oxide also gave rise to DNA adducts in rat liver cells, but the adduct patterns were very similar to those formed by tamoxifen and alpha-hydroxytamoxifen, indicating that the N-oxygen is lost prior to DNA binding. These and earlier results demonstrate that in rat liver cells in vivo and in vitro, Phase I metabolic activation of tamoxifen involves both alpha-hydroxylation and N-demethylation, which is followed by Phase II activation at the alpha-position to form a highly reactive sulphate. Detection of tamoxifen-related DNA adducts by (32)P-postlabelling is achieved with >90% labelling efficiency.

Idoxifene derivatives are less reactive to DNA than tamoxifen derivatives, both chemically and in human and rat liver cells.

The drug tamoxifen shows evidence of genotoxicity, and induces liver tumours in rats. Covalent DNA adducts have been detected in the liver of rats treated with tamoxifen, and these arise through metabolism at the alpha-position to give an ester which reacts with DNA. (E)-1-(4-iodophenyl)-2-phenyl-1-[4-(2-pyrrolidinoethoxy)phenyl]-but-1-en e (idoxifene) is an analogue of tamoxifen in which formation of DNA adducts is greatly reduced; we could not detect any adducts in the DNA of cultured rat hepatocytes treated with 10 microM idoxifene, after analysis by the 32P-post-labelling method. The metabolite (Z)-4-(4-iodophenyl)-4-[4-(2-pyrrolidinoethoxy)phenyl]-3-phenyl-3-but en-2-ol (alpha-hydroxyidoxifene) gave adducts in rat hepatocytes, but far fewer than the corresponding tamoxifen metabolite. In human hepatocytes, neither idoxifene nor tamoxifen induced detectable levels of DNA adducts. We prepared the alpha-acetoxy ester of idoxifene as a model for the ultimate reactive metabolite formed in rat liver. It was less reactive than alpha-acetoxytamoxifen, as might be expected on mechanistic grounds. It reacted with DNA in the same way, to give adducts which were probably N2-alkyldeoxyguanosines, but to a lower extent. All these results indicate that idoxifene is much less genotoxic than tamoxifen, and should therefore be a safer drug.

4-Hydroxytamoxifen gives DNA adducts by chemical activation, but not in rat liver cells.

The drug tamoxifen shows evidence of genotoxicity, and induces liver tumors in rats. Covalent DNA adducts have been detected in the liver of rats treated with tamoxifen, and in rat hepatocytes in culture. These arise primarily from its metabolite alpha-hydroxytamoxifen, and may also arise, in part, from another metabolite, 4-hydroxytamoxifen. We have prepared two model compounds for the potential reactive metabolite formed from 4-hydroxytamoxifen in rat liver. One of these was its alpha-acetoxy ester. This was much more reactive than that from tamoxifen, and could not be isolated in pure form. It reacted with DNA in the same way that alpha-acetoxytamoxifen did, to give adducts which were isolated by hydrolysis and chromatography, and identified as alkyldeoxyguanosines. The second derivative was alpha, beta-dehydro-4-hydroxytamoxifen. This also reacts with DNA in vitro, to give the same products as those from alpha-acetoxy-4-hydroxytamoxifen. Reaction probably proceeds through the same resonance-stabilized carbocation in either case. However, when primary cultures of rat hepatocytes were treated with either 4-hydroxytamoxifen, 4,alpha-dihydroxytamoxifen, or alpha, beta-dehydro-4-hydroxytamoxifen at a concentration of 10 microM, no adducts could be detected in their DNA by the 32P-postlabeling technique. Similarly, no adducts could be found in the liver DNA of female Fischer F344 rats treated orally (at 0.12 mmol kg-1) with the same substances. If 4-hydroxytamoxifen is metabolized to 4, alpha-dihydroxytamoxifen in rat liver, then either this substance is not converted to reactive esters or they are rapidly detoxified.

Synthesis and DNA reactivity of alpha-hydroxylated metabolites of nonsteroidal antiestrogens.

Tamoxifen [(E)-1-(4-(2-(N,N-dimethylamino)ethoxy)phenyl)-1, 2-diphenylbut-1-ene], a nonsteroidal antiestrogen, induces liver tumors in rats by a genotoxic mechanism. The mechanism of DNA adduct formation is believed to proceed via the formation of a reactive carbocation at the alpha-position from the alpha-hydroxylated metabolite. Molecular mechanics calculations [Kuramochi, H. (1996) J. Med. Chem. 39, 2877-2886] have predicted that 4-substitution will affect the stability of the carbocation and thus will alter its reactivity toward DNA. We have synthesized the putative alpha-hydroxylated metabolites of 4-hydroxytamoxifen [(E)-1-(4-(2-(N, N-dimethylamino)ethoxy)phenyl)-1-(4-hydroxyphenyl)-3-hydroxy-2-phenyl but-1-ene] and idoxifene [(Z)-1-(4-iodophenyl)-3-hydroxy-2-phenyl-1-(4-(2-(N-pyrrolidino) ethoxy)phenyl)but-1-ene] and compared their reactivities with DNA with that of alpha-hydroxytamoxifen [(E)-1-(4-(2-(N, N-dimethylamino)ethoxy)phenyl)-3-hydroxy-1,2-diphenylbut-1-ene]. As predicted, the bis-hydroxylated compound reacted with DNA in aqueous solution at pH 5 to give 12-fold greater levels of adducts than alpha-hydroxytamoxifen, whereas alpha-hydroxyidoxifene gave one-half the number of adducts. The results demonstrate that idoxifene presents a significantly lower genotoxic hazard than tamoxifen for the treatment and prophylaxis of breast cancer.

Minor products of reaction of DNA with alpha-acetoxytamoxifen.

The drug tamoxifen shows evidence of genotoxicity and induces liver tumours in rats. Covalent DNA adducts have been detected in the liver of rats treated with tamoxifen and these arise, at least in part, from its metabolite alpha-hydroxytamoxifen. This probably undergoes conjugation in the liver tissue to give an ester, which alkylates DNA. We have prepared alpha-acetoxytamoxifen as a model for this reactive intermediate and studied its reaction with DNA in vitro. The products of this reaction were chromatographically identical to DNA adducts found in the liver of rats treated with tamoxifen. We have isolated three of these products as the nucleosides TG1, TG2 and TA1 and identified them by ultraviolet, mass and proton magnetic resonance spectroscopy. TG1 and TG2 were tamoxifen-deoxyguanosine adducts in which the alpha-position of tamoxifen was linked to the amino group of guanine; TG1, (E)-4-[4-[2-(dimethylamino)ethoxy]phenyl]-3,4-diphenyl-2-(9beta-de oxyribofuranosyl-6-oxopurin-2-ylamino)-3-butene; TG2, (Z) isomer of TG1. In TG2, the tamoxifen group had undergone trans-cis isomerization. The minor product TA1 was a tamoxifen-deoxyadenosine adduct, where linkage was through the amino group of adenine: (E)-4-[4-[2-(dimethylamino) ethoxy]phenyl]-3,4-diphenyl-2-(9beta-deoxyribofuranosylpurin -6-ylamino)-3-butene. These three adducts accounted for >90% of the reaction products (approximately 67% TG1, 18% TG2 and 7% TA1); trace products included other stereoisomers of these and dinucleotide adducts which resisted enzymatic digestion.

Studies of the binding of diolepoxide metabolites of polycyclic aromatic hydrocarbons to DNA using electrofluorescence polarization spectroscopy.

In the electrofluorescence method, a solution of DNA with covalently bound polycyclic hydrocarbons is placed in an electric field, and changes in the intensity of polarized fluorescence are observed. Under the correct conditions, these charges can be used to determine a value for the angle psi between the long axis of the hydrocarbon molecule and the axis of the DNA helix. For DNA or poly(dA-dT) treated with each stereoisomer of anti-benzo[c]phenanthrene diolepoxide, psi ranged from 55 degrees to 61 degrees, consistent with a mixture of quasi-intercalated adenine adducts and externally bound guanine adducts. Similar results were obtained with another set of 'fjord-region' diolepoxides, derived from benzo[c]chrysene. Adducts in DNA treated with diolepoxides derived from chrysene, 5-methylchrysene or 6-methylchrysene gave psi of about 53 degrees, so the predominant adducts are externally bound, probably in the minor groove of DNA.

Identification of the major tamoxifen-deoxyguanosine adduct formed in the liver DNA of rats treated with tamoxifen.

The antiestrogenic drug tamoxifen induces liver tumors in rats by a genotoxic mechanism. The key step has been proposed to be the formation of a reactive carbocation from the metabolite alpha-hydroxytamoxifen. This compound reacts with DNA in vitro to a small extent (1 in 10(5) DNA bases), giving products identical to those found in rat liver cells treated with tamoxifen. Now we have prepared the more reactive alpha-acetoxytamoxifen, which reacts with DNA in vitro to a much greater extent (1 in 50 bases). The products of this reaction were subjected to 32P postlabeling and shown by both TLC and reverse-phase liquid chromatography to be identical to those isolated from DNA treated with alpha-hydroxytamoxifen and to those found in the liver DNA of rat hepatocytes treated with tamoxifen or of the livers of rats treated with tamoxifen. The major product was also isolated as the nucleoside and characterized by UV, mass, and proton magnetic resonance spectroscopy. It is an adduct of tamoxifen and deoxyguanosine in which the alpha position of tamoxifen is linked covalently to the exocyclic amino group of deoxyguanosine.

Products from alkylation of DNA in cells by melphalan: human soft tissue sarcoma cell line RD and Escherichia coli WP2.

Alkylation of DNA was studied after treatment with [3H]melphalan (phenylalanine mustard; 1-2 microM) using a human tumour cell line, RD, in culture, or Escherichia coli (WP2 or WP2-uvrA strains) in growth medium. After 6 h at 37 degrees C, treated cells were isolated and re-suspended in fresh growth media. Samples were taken at times up to 48 h for isolation of DNA, and in some cases also RNA and protein (which were found to be alkylated to about the same extent as DNA). Alkylated DNA was analysed as previously described (M.R. Osborne and P.D. Lawley, Chem.-Biol. Interact 89 (1993) 49-60). The four principal products, mono-7-alkylguanine (G-M-OH); mono-3-alkyladenine (A-M-OH); and the cross-linked products G-M-G and A-M-G, were identified in DNA from melphalan treated cells, and quantitatively determined. In each case, alkylation of cellular macromolecules was maximal after about 6 h. In DNA of the human tumour cell line, the relative amounts of adenine products decreased with time, most markedly with A-M-OH to 42% of the 2-h value after 48 h. In DNA of both bacterial strains, A-M-OH was virtually undetectable even at early times. Comparisons between the time course of relative decreases in amounts of these alkylpurine products and the corresponding values for alkylated DNA in vitro suggest that the adenine products are subject to removal by repair enzyme action in E. coli of either strain.

Alkylation of DNA by the nitrogen mustard bis(2-chloroethyl)methylamine.

Alkylation of DNA by the nitrogen mustard bis(2-chloroethyl)methylamine (mechlorethamine; HN2) gave four principal products, derived by mono-alkylation of guanine at N-7 and adenine at N-3 and by cross-linking of guanine to guanine or guanine to adenine at these positions. These products were isolated by hydrolysis from DNA at neutral pH, followed by ion-exchange chromatography on SP-Sephadex and reversed phase chromatography on ODS. They were characterized by identification with products from the reaction of nitrogen mustard with adenine or deoxyguanylic acid, and by their UV, mass, and proton magnetic resonance spectra.

Detection of bulky DNA lesions in the liver of patients with Wilson's disease and primary haemochromatosis.

In the human metal storage disorders of Wilson's disease and primary haemochromatosis, ion transport and excretion dysfunctions result in the intracellular deposition of copper and iron, respectively. These aberrant accumulations of transition metal ions lead to extensive tissue damage, especially in the liver. In order to investigate the possible role of metal ion-mediated oxygen free radical-generated DNA damage in these processes, DNA was isolated from liver of eight Wilson's disease patients and six haemochromatosis patients. Significant levels of bulky DNA damage were detected in these samples by 32P-postlabelling analysis, but were not found in liver DNA from age-matched controls. This form of novel DNA damage was detected in six out of eight Wilson's patients, varying between approximately 1 and 100 base modifications per 10(8) nucleotides, and in all of the haemochromatosis samples examined; the levels of modified species per 10(8) nucleotides varying from approximately 2 to 50. HPLC analysis of these bulky DNA lesions demonstrated that the species formed in Wilson's disease and in haemochromatosis were chromatographically identical but were not the same as putative purine dimers that can be generated in DNA by in vitro incubation with Cu+/Fe2+ and H2O2 (although the possibility that the adducts detected are closely related has not been ruled out). Analysis of the oxidative base lesion 8-hydroxydeoxyguanosine showed that levels were not elevated in liver DNA from either Wilson's disease or haemochromatosis sufferers. In fact, a statistically significantly lower level of this lesion was found in Wilson's disease patients than in controls. These data suggest that bulky DNA damage present in the liver of both wilson's disease and primary haemochromatosis patients may play a more important role in the induction of tissue damage than 8-hydroxydeoxyguanosine. The novel DNA damage detected by 32P-poslabelling may also be a significant factor in the initiation of neoplasia leading to malignant hepatoma in haemochromatosis patients.

Multikilohertz stimulated Brillouin scattering.

We report, for the first time to our knowledge, multikilohertz stimulated-Brillouin-scattering phase conjugation of copper-vapor laser radiation. The small wavelength shift associated with the Stokes radiation is shown to permit double-pass aberration correction of the copper-vapor laser, avoiding the postlasing absorption. Preliminary results on the effects of stimulated-Brillouin-scattering medium type, purity, geometry, and flow on the efficiency are presented, as is an initial assessment of the conjugation fidelity.

Alkylation of DNA by melphalan with special reference to adenine derivatives and adenine-guanine cross-linking.

Alkylation of DNA by melphalan gave four principal products, derived by mono-alkylation of adenine at N-3 and guanine at N-7, and by cross-linking of adenine N-3 to guanine N-7, or of guanine N-7 to guanine N-7. Adenine-guanine cross-linking was unexpected because the two principal nucleophilic centres, N-7 of guanine and N-3 of adenine, are situated in the 'wide' and 'narrow' grooves of the DNA double helix, respectively. These products could be isolated by their hydrolysis from DNA at neutral pH, followed by chromatography of the hydrolysate in an ion-pair ODS system using a solvent containing tetrabutylammonium hydroxide as ion-pairing reagent; this gave better separation than the previously described method using SP-Sephadex.

The reaction of melphalan with deoxyguanosine and deoxyguanylic acid.

The reaction of melphalan (phenylalanine mustard, I) with 2'-deoxyguanosine, followed by removal of the sugar in acid, yielded two products. The major product was identified as 4-(N-(2-guanin-7-ylethyl)-N-(2-hydroxyethyl)amino)phenyl- alanine (II) by ultra-violet absorption, mass and NMR spectroscopy. The minor product has already been identified as the corresponding bis-guaninyl adduct III (Tilby et al., Chem.-Biol. Interact., 73 (1990) 183-194). The reaction of melphalan with 5'-deoxyguanylic acid yielded the deoxyribonucleotide of II and products resulting from reaction with the phosphate group. The initial products, which were formed with a half-life of approximately 40 min at 37 degrees, still had a reactive chloroethyl group; this was displaced more slowly, by reaction with water or with another molecule of dGMP. The products of reaction of melphalan with DNA were released by treatment with acid (0.1 M HCl, 70 degrees, 30 min) and separated from each other on a cation exchange column. They were identified as II, III and an adenine adduct, in a ratio of approximately 3:1:2.

Interaction of omeprazole with DNA in rat tissues.

There has been concern recently over the possible genotoxicity of omeprazole, a potent inhibitor of gastric acid secretion in humans. In order to investigate its possible DNA binding activity in vivo, 14C-labelled omeprazole was administered to male PVG rats, 8-10 weeks old, orally by gavage at a dose of 30 mg/kg. At various times after treatment, animals were killed and DNA isolated from the fundic and antral regions of the glandular stomach, and from the duodenum, ileum and colon. Scintillation counting of the DNA samples revealed a reproducible association of 14C that was not extractable with organic solvents and could not be removed from the DNA by column cartridge chromatography. DNA samples were digested enzymically to deoxyribonucleosides and analysed by reversed-phase high performance liquid chromatography (HPLC). With DNA from each tissue, most of the radioactivity eluted in a peak that was chromatographically distinct from normal nucleosides and from omeprazole itself. Chromatography following partial digestion of DNA gave rise to the same radioactivity elution profiles, whilst chromatography on hydroxyapatite resulted in retention of most of the radioactivity on the column using conditions under which DNA was eluted. These findings suggest either the formation in rat tissues of chemically-labile covalent omeprazole-DNA adducts or, more likely, a strong non-covalent interaction. The apparent DNA binding occurs rapidly in vivo and is short-lived, the maximum levels of radioactive incorporation (equivalent of up to 36 pmol omeprazole/mg DNA) occurring at 0.5 h in fundus, 0.5-1 h in antrum, 1 h in duodenum, 2 h in ileum and at 4-8 h in colon.

Comeditation: an exploratory study of pulse and respiration rates and anxiety.

To achieve deep relaxation in seriously ill persons, Tibetan medicine has employed a breathing process, known as "comeditation," which requires a caregiver to focus attention on the chest of the reclining patient while making a sound or number keyed to the patient's exhalation. This study investigated the relationship between state and trait anxiety and lowered respiratory rate, using the comeditation procedure. Ten subjects were assigned randomly to either a control or comeditation group. Anxiety was measured on the State-Trait Anxiety Inventory. Analysis indicated a decrease in State-Anxiety scores in the comeditation group, but no differences between groups in pulse and respiration rates or trait anxiety. Implications for theory and research are discussed.

Sequence specificity in the reaction of benzopyrene diol epoxide with DNA.

Benzopyrene diol epoxide (BPDE; (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-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 labelled 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.