Non-covalent intercalative binding of 7,8-dihydroxy-9,10-epoxybenzo(a)pyrene to DNA

When the benzo(a)pyrene diol epoxide (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) is mixed into a DNA solution, a 10nm red shift in the absorption maximum of BPDE appears at 354nm which is due to a non-covalent intercalation complex. The major reaction pathway at this intercalation site is the hydrolysis of BPDE to its tetraol which is accompanied by a decrease in the absorbance and a shift from 354 to 353nm (the latter is due to intercalated tetraol). The non-covalent binding constants are approximately 8200M^?1 for BPDE and 3300M^?1 for the tetraol at 25°C, pH 7.0. Covalent adduct formation between BPDE and DNA occurs either at another, external binding site, or after some rearrangement of the intercalated BPDE, since covalent adducts display a 345nm absorption maximum (2nm red shift only).     

Noncovalent intercalative complex formation and kinetic flow linear dichroism of racemic syn- and anti-benzo[a]pyrenediol epoxide-DNA solutions

In order to gain a better understanding of the molecular basis underlying the differences in biological activities of the diastereomeric syn and anti diol epoxides of benzo[a]pyrene (trans-7,8-dihydroxy-syn-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, respectively), their interactions with DNA in aqueous solutions were studied and compared. Kinetic flow linear dichroism experiments indicate that both diastereomers (racemic mixtures) form intercalation complexes immediately after mixing; the association constant (23 degrees C, ionic strength approximately 0.005) is significantly smaller (5200 M-1) in the case of the syn than in the case of the anti diastereomer (12 200 M-1). This difference is attributed to the greater bulkiness of the 7,8,9,10 ring of the syn stereoisomer, which is in the quasi-diaxial conformation as compared to the less bulky quasi-diequatorial conformation of the anti diastereomer. In contrast, the intercalative association constants of the tetraols derived from the hydrolysis of the two diol epoxides are similar in value. Upon formation of noncovalent syn-BPDE-DNA intercalation complexes, the reaction rate constant for the formation of tetraols (approximately 98%) and covalent adducts (approximately 2%) increases from 0.009 to 0.05 s-1 at pH 9.5 in 5 mM tris(hydroxymethyl)aminomethane buffer. The conformations of the aromatic chromophores of BPDE were followed by the kinetic flow dichroism technique as a function of reaction time; while the anti diastereomer changes conformation from an intercalative to an apparently external binding site, the syn diol epoxide molecules do not appear to undergo any measurable reorientation during or after the covalent binding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural effects in reactivity and adduct formation of polycyclic aromatic epoxide and diol epoxide derivatives with DNA: comparison between 1-oxiranylpyrene and benzo[a]pyrenediol epoxide

Reaction of 1-oxiranylpyrene (1-OP) with DNA and the structures of the covalent and noncovalent complexes formed were studied in aqueous media (5 mM phosphate buffer with 0.1 M NaCl, pH 7) by utilizing the techniques of absorption, fluorescence and linear dichroism spectroscopy in order to gain an understanding of possible structure-activity relationships for polycyclic aromatic hydrocarbon epoxides in tumorigenesis and carcinogenesis, and the results were compared with those obtained for the highly active benzo[a]pyrene diol epoxide (BaPDE). Like BaPDE, 1-OP undergoes acid-catalyzed hydrolysis with the pseudo-first-order rate constant k = 4.6 X 10(-4) s-1 in the absence of DNA, which is about 10 times slower than in the case of BaPDE. In DNA solutions, this hydrolysis is catalyzed by a rapid formation of a physically bound complex of 1-OP-DNA, which subsequently undergoes either (1) hydrolysis to a diol derivative or (2) formation of a covalent adduct of 1-OP-DNA. The same value of the noncovalent binding constant (K = 4000 M-1 is obtained for both 1-OP and for BaPDE, which suggests that the pi-electron interaction between the pyrenyl moiety and the nucleic acid bases is the dominant factor in the formation of the physical complexes and that the two extra OH groups in BaPDE do not play a significant role in determining the value of the physical binding constant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carcinogen-induced alteration of DNA structure

We have prepared covalent complexes between defined length DNA fragments and a diol epoxide derivative of the carcinogenic polycyclic aromatic hydrocarbon, benzo(a)pyrene. We have studied the structure of these complexes, using transient electric dichroism, circular dichroism, fluorescence quenching, thermal denaturation, polyacrylamide gel electrophoresis, and nuclease digestion. Our observations suggest that th covalently bound carcinogen is intercalated within the helix, forming a wedge-shaped complex. Binding of the carcinogen distorts the structure of the DNA over a region extending beyond the immediate binding site. The most striking aspect of this distortion is that it produces a bend in the helix.     

Repair of DNA damaged by mutagenic metabolites of benzo(a)pyrene in human cells

The repair of human DNA after damage by known and potential metabolites of benzo(a)pyrene has been examined utilizing the bromodeoxyuridine photolysis assay. Repair was characterized as either ultraviolet (“long”) or ionizing radiation type (“short”) repair utilizing normal cells and cells deficient in ultraviolet-type repair endonuclease from a patient with xeroderma pigmentosum (XP). We have found that only (±)-7β,8-dihydroxy-9β,-10β-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BP diol epoxide 1) and its disastereomer, (±)-7β,8,-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BP diol epoxide 2) elicit damage to DNA which is recognizable by the ultraviolet excision repair system in normal human cells. Benzo(a)pyrene 4,5-, 9,10-, 11,12-oxides do not elicit damage which is repairable by this repair system. The 1,2-diol-3,4-epoxides from naphthalene have no measurable activity in our assay. These results indicate that both the benzo(a)pyrene ring structure and the diol epoxide groups are important in causing the damage to DNA which is repairable by the ultraviolet excision repair system. These results parallel the reported high mutagenic activity of these compounds and support the concept that benzo(a)pyrene 7,8-diol-9,10-epoxides may be the ultimate, metabolically activated forms of benzo(a)pyrene.     

Effect of ellagic and caffeic acids on covalent binding of benzo[a]pyrene to epidermal DNA of mouse skin in organ culture

The covalent binding of the anti-diol epoxide of benzo[a]pyrene to cellular DNA of mouse skin in organ culture is affected by the presence of ellagic acid in the culture medium. At 10(-4) M, BaPDE /DNA formation is 40% less than that observed when no ellagic acid is present. Caffeic acid, a similar plant phenolic compound, demonstrates no inhibitory effect on BaPDE /formation. The plant phenolic acids do not drastically interfere with the metabolism of benzo[a]pyrene as shown by the BaP-metabolite profiles of the skin or of the culture medium.     

Kinetics of hydrolysis to tetraols and binding of benzo(a)pyrene-7,8-dihydrodiol-9,10-oxide and its tetraol derivatives to DNA. Conformation of adducts

When the major reactive metabolite of benzo(a)pyrene, $\text{trans}$ -7,8-dihydroxy - $\text{anti}$-9,10-epoxy -7,8,9,10-tetrahydrobenzo(a)pyrene ($\text{anti}$-BPDE) is incubated with DNA in aqueous solution at 25°C, both covalent binding and hydrolysis of $\text{anti}$-BPDE to its tetraols occur. Using fluorescence and absorption spectroscopy it is shown that hydrolysis of $\text{anti}$-BPDE is markedly accelerated by DNA. In the presence of 5A₂₆₀ units of DNA per ml in cacodylate buffer solution, at an initial concentration of DNA phosphate/$\text{anti}$-BPDE ratio of 100, both the extent of covalent binding to DNA ( < 7% of the total $\text{anti}$-BPDE initially present) and hydrolysis of $\text{anti}$-BPDE reach their maximum levels within less than five minutes after mixing. Absorption and electric linear dichroism spectra indicate that the tetraols bind non-covalently to DNA by an intercalation mechanism, whereas the covalent product displays the characteristics of an externally bound complex.     

Ethoxyquin feeding to rats increases liver microsome-catalyzed formation of benzo(a)pyrene diol epoxide — DNA adduct

The ability of rat liver microsomes to catalyze the formation of benzo(a)pyrene 7,8-diol-9,10-epoxide — DNA nucleoside adduct was increased threefold by feeding 0.5% ethoxyquin to the animals. Microsomal epoxide hydratase activity was enhanced i parallel by a factor of 3 while aryl hydrocarbon hydroxylase activity was not induced. Liver microsomes from rat pretreated with 3-methylcholanthrene produced an increased proportion of diol epoxide — DNA adduct when ethoxyquin had been fed to the animals. The main chromatographic peak formed by microsomes from 3-methylcholanthrene treated rats which contains DNA adducts of secondary benzo(a)pyrene phenol metabolites is reduced when the animals had received ethoxyquin.     

Reactions of stereoisomeric and structurally related bay region diol epoxide derivatives of benz[a]anthracene with DNA. Conformations of noncovalent complexes and covalent carcinogen-DNA adducts

The modes of reaction of the tumorigenic bay region diol epoxide anti-BADE [+/-)-trans-3,4-diol-anti-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthr acene) and the less potent tumor initiating diastereomer syn-BADE [+/-)-trans-3,4-diol-syn-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthra cene) with native, double-stranded DNA were compared. The bay-region diol epoxide derived from 3-methylcholanthrene (3-MCDE, racemic trans-9,10-diol-anti-7,8-epoxy-7,8,9,10-tetrahydromethylcholanthrene+ ++) was included in this study in order to assess the effects of the methyl and methylene substituents on the reactivity with DNA. Utilizing linear dichroism and other spectroscopic methods, it is shown that all three diol epoxides forn non-covalent complexes with DNA. The diastereomers anti-BADE and syn-BADE form intercalative physical complexes, but the association constant K of the syn-diastereomer is about 6-7 times smaller than for anti-BADE; this effect is ascribed to the bulky quasi-diaxial conformation of the diol epoxide ring in the syn diastereomer. The value of K (4000 M-1) is similar for anti-BADE and 3-MCDE, although the latter is not intercalated in the classical sense since the short axis of the molecule is tilted closer to the axis of the DNA double helix. The conformations of the covalent DNA adducts are interpreted in terms of a quasi-intercalative conformation (site I), and a conformation in which the long axes of the polycyclic molecules are tilted closer to the axis of the helix (site II). Both tumorigens, anti-BADE and 3-MCDE, undergo a marked re-orientation from a non-covalent site I to a covalent site II conformation upon binding chemically with the DNA bases, although a small fraction of the covalent anti-BADE adducts remains quasi-intercalated; in contrast, the alkyl substituents in 3-MCDE not only prevent the formation of intercalative physical complexes, but also the formation of site I covalent adducts. In the case of the less tumorigenic syn-BADE, both the non-covalent complexes and the covalent adducts are of the site I-type. The bay-region diol epoxide of benz[a]anthracene and of 3-methylcholanthrene display a similar pattern of reactivities and covalent adduct conformations as the bay region diol epoxide derivatives of benz[a]pyrene, suggesting that adduct conformation might be an important factor in determining the levels of mutagenic and tumorigenic activities of this class of compounds.     

Mechanisms of reaction of benzo(a)pyrene-7,8-diol-9,10-epoxide with DNA in aqueous solutions

The physical and chemical reaction pathways of the metabolite model compound benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) in aqueous (double-stranded) DNA solutions was investigated as a function of temperature (0-30 degrees C), pH (7.0-9.5), sodium chloride concentration (0-1.5M) and DNA concentration in order to clarify the relationships between the multiple reaction mechanisms of this diol epoxide in the presence of nucleic acids. The reaction pathways are (1) noncovalent intercalative complex formation with DNA, characterized by the equilibrium constant K, and Xb the fraction of molecules physically bound; (2) accelerated hydrolysis of BPDE bound to DNA; (3) covalent binding to DNA; and (4) hydrolysis of free BPDE(kh). The DNA-induced hydrolysis of BPDE to tetraols and the covalent binding to DNA are parallel pseudo-first-order reactions. Following the rapid (millisecond time scale) noncovalent complex formation between BPDE and DNA, a much slower (approximately minutes) H+-dependent (either specific or general acid catalysis) formation of a DNA-bound triol carbonium ion (rate constant k3) occurs. At pH 7.0 the activation energy of k3 is 8.7 +/- 0.9 kcal/mol, which is lower than the activation energy of hydrolysis of free BPDE in buffer solution (14.2 +/- 0.7 kcal/mol), and which thus partially accounts for the acceleration of hydrolysis of BPDE upon complexation with DNA. The formation of the triol carbonium ion is followed by a rapid reaction with either water to form tetraols (rate constant kT), or covalent binding to DNA (kc). The fraction of BPDE molecules which undergo covalent binding is fcov approximately equal to kc/(kc + kT) = 0.10 and is independent of the overall BPDE reaction rate constant k = kh(1 - Xb) + k3Xb if Xb----1.0, or is independent of Xb as long as k3Xb much greater than kh(1 - Xb). Thus, at Xb = 0.9, fcov is independent of pH (7.0-9.5) even though k exhibits a 70-fold variation in this pH range and k----kh above pH 9 (k3 = kh). Similarly, fcov is independent of temperature (0-30 degrees C), while k varies by a factor of approx. 3. In the range of 0-1.5 M NaCl, fcov decreases from 0.10 to 0.04. These variations are attributed to a combination of salt-induced variations in the factors k3, Xb and the ratio kc/kT.     

Binding geometries of benzo[a]pyrene diol epoxide isomers covalently bound to DNA. Orientational distribution

Flow linear dichroism (LD) of different benzo[a]pyrene diol epoxide (BPDE) isomers covalently bound to calf thymus DNA or poly(dG-dC) provides information about binding geometry and DNA perturbation. With anti-BPDE the apparent angle between the long axis (z) of the pyrene chromophore and the DNA helix axis is approximately 30 degrees as evidenced from the LD of z-polarized absorption bands in the pyrenyl chromophore at 252 and 346 nm. The corresponding angle for the in-plane short axis (y) is determined to be approximately 70 degrees from a y-polarized band at 275 nm. The binding of (+)-anti-BPDE to DNA is found to cause a considerable reduction of the DNA orientation. This is ascribed to a decreased persistence length of DNA, owing either to increased flexibility ("flexible joints") or to permanent kinks at the points of binding. The reduced linear dichroism (LDr), i.e., the ratio between LD and isotropic absorbance, of the long-wavelength absorption band system of BPDE bound to DNA exhibits a wavelength dependence that indicates a relatively wide orientational distribution of the z axis of pyrene. Fluorescence data support the conclusion of a heterogeneous distribution, and a very low polarization anisotropy indicates a mobility between the different orientational states, which is rapid compared to the fluorescence lifetime (nanosecond time scale). Attempts are made to simulate the observed LDr features of the (+)-anti-BPDE-poly(dG-dC) complex using different distribution models on the assumption that the angular dependence of the spectral perturbation is due to dispersive interactions with DNA bases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of stability of 7 beta,8 alpha-dihyroxy-9 alpha epoxybenzo(a)pyrene by complex formation with serum albumin

The biologically active and chemically unstable metabolite of benzo(a)pyrene, 7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxybenzo(a)pyrene (BPDE), binds to human serum albumin in aqueous solutions with an association constant of 2.6 X 10(5)M-1. At pH 7.2, 24 degrees C and in 5mM sodium cacodylate buffer solution, this binding increases the lifetime of the diol epoxide by a factor of nearly 3. It is suggested that the formation of such physical complexes with proteins having hydrophobic interiors or with lipids may provide a mechanism by which highly reactive metabolites are transported from the site of metabolic synthesis to biological target molecules (e.g., DNA), in a reactive aqueous environment.     

Effect of purified epoxide hydrolase on metabolic activa-tion and binding of benzo(a)pyrene to exogenous DNA. Shift of the activation pathway

The effect of purified epoxide hydrolase (E.C. 3.3.2.3) on the binding of benzo(a)pyrene metabolites 9-hydroxybenzo(a)pyrene and 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene to DNA catalyzed by cytochrome P 448 from liver microsomes of methylcholanthrene pretreated rats has been investigated. The total binding and the major binding species derived from 9-hydroxybenzo(a)pyrene were strongly inhibited by the presence of purified epoxide hydrolase and the species derived from 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene was slightly increased. By modifying the balance between cytochrome P 448 and epoxide hydrolase it is possible to shift quantitatively the binding of these two main reactive intermediates to DNA.     

Catalysis of carcinogen-detoxification by DNA: comparison of enantiomeric diol epoxides

The interactions of the (+)- and (-)-enantiomers of 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I) with purified DNA have been studied in vitro. These compounds are formed by cellular metabolism of the potent environmental carcinogen benzo[a]pyrene, and the (+)-enantiomer is thought to be the ultimate carcinogenic metabolite. Non-covalent, intercalative binding was measured spectrophotometrically, hydrolysis was measured spectrofluorometrically and covalent binding was detected by liquid scintillation counting. No significant differences were found in the association constants for intercalative binding or in the ability of DNA to catalyse the hydrolysis of the two enantiomers. Covalent DNA binding was 4.5-fold higher for the (+)-enantiomer. When DNA was pretreated with a molar equivalent of the (-)-enantiomer, its subsequent ability to enhance the rate of BPDE-I hydrolysis and to bind covalently to (+)-BPDE-I was unimpaired. This suggests that the participation of the DNA in the hydrolysis reaction does not alter the DNA and therefore that the rate-enhancement is true catalysis.     

Kinetics of DNA alkylation, depurination and hydrolysis of anti diol epoxide of benzo(a)pyrene and the effect of cadmium on DNA alkylation

Anti benzo[a]pyrene diol epoxide (BPDE) alkylates guanines of DNA at N7 in the major groove and at the exocyclic amino group in the minor groove. In this report we investigated the rates of BPDE hydrolysis, DNA alkylation and subsequent depurination of BPDE-adducted pBR322 DNA fragment using polyacrylamide gel electrophoresis. Preincubation studies showed that it hydrolyzed completely in triethanolamine buffer in <2 min. The depurination kinetics showed that a fraction of the N7 alkylated guanine depurinated rapidly; however a significant amount of N7 guanine alkylation remained stable to spontaneous depurination over a 4-h period. Similar results were obtained for the hydrolysis and alkylation rates of syn isomer but it required nearly 500 times more concentration to induce similar levels of N7 guanine alkylation. Cadmium ion strongly inhibited the N7 guanine alkylation of both isomers. But the minor groove alkylation was not affected as demonstrated by postlabeling assay which confirmed the presence of heat-and cadmium-stable minor groove adducts in BPDE-treated calf thymus DNA. Based on these and our earlier findings, we propose a mechanism for the synergistic effect of cadmium in chemically induced carcinogenesis.     

The conversion of benzo(alpha)pyrene 4,5-oxide into 4-hydroxybenzo(alpha)pyrene in the presence of polyriboguanylic acid.

Incubation of benzo[alpha] pyrene 4,5-oxide with poly(G) in neutral aqueous ethanol resulted in the formation of covalent adducts and in the production of free 4-hydroxybenzo[alpha]pyrene. This phenol, which was identified by its UV spectral properties and by its chromatographic characteristics, was also formed but at a much slower rate when the epoxide was incubated with DNA or with GMP. Phenol formation was not detected when benzo[alpha]-pyrene 4,5-oxide was incubated for prolonged periods in the presence of poly(A), poly(C) or poly(U) or in the absence of nucleic acid. Formation of 4-hydroxybenzo[alpha] pyrene from the epoxide in the presence of poly(G) was not accompanied by detectable base modifications or by breakage of phosphodiester linkages.     

Magnesium ions affect the quantitative but not the qualitative microsome mediated binding of benzo[alpha]-pyrene to DNA

Five distinct hydrocarbon-deoxyribonucleoside adducts are separated by high pressure liquid chromatography after reaction of benzo[alpha]pyrene with calf thymus DNA in the presence of liver microsomes from 3-methylcholanthrene treated rats. The two major adducts co-chromatography with deoxyribonucleoside adducts obtained after hydrolysis of calf thymus DNA previously reacted with liver microsomal metabolically activated 9-hydroxy-benzo[alpha]pyrene or trans-7,8-dihydro-7,8-dihydroxybenzo[alpha]pyrene. High magnesium ion concentrations in the microsomal incubations cause a significant decrease in the covalent binding of the hydrocarbon to DNA but do not affect the qualitative distribution of the individual benzo[alpha]pyrene-deoxyribonucleoside adducts.     

In vivo benzo[a]pyrene diol epoxide-induced alkali-labile sites are not apurinic sites.

We have used endonuclease IV from Escherichia coli as a probe for apurinic sites in the DNA of HeLa cells following treatment with an activated diol epoxide derivative of benzo[a]pyrene. DNA strand breaks and alkali-labile sites were observed that were repaired following exposure to the carcinogenic alkylating agent. The alkali-labile sites were not substrates for the apurinic site-specific endonuclease IV. We conclude that the alkali-labile sites formed in vivo by benzo[a]pyrene derivatives are not apurinic sites and probably arise as a consequence of rearrangement of the abundant N2-guanine adducts. This finding questions the involvement of apurinic sites in the mutagenic activity of benzo[a]pyrene.     

Equilibrium binding of derivatives of the carcinogen, benzo(a)pyrene, to DNA. Thermodynamic analysis

The physical binding of polycyclic aromatic hydrocarbon derivatives which are ultimate carcinogens to DNA may play a role in the formation of covalent DNA adducts by these compounds or in the detoxification of the compounds via DNA-catalyzed hydrolysis. Previous studies of DNA-binding interactions of derivatives of benzo(a)pyrene (BP) have been confined to low r values (r - ligands bound/base pair). We have now applied the Scatchard formalism (as modified to include neighbor exclusion) to the spectrophotometric determination of the binding of two derivatives of BP, trans - 9,10 - dihydroxydihydro - BP and 7r,8t - dihydroxy-9t,10t-oxy-7,8, 9,10-tetrahydro-BP, to double-stranded DNA at reasonably high r values. Exclusion parameters, binding constants, and thermodynamic parameters are all within the ranges found for other intercalants. Although these ligands are uncharged, the binding exhibits significant ionic strength dependence which can be rationalized (partially) by polyelectrolyte theory. Using the measured ionic strength dependence, a thermodynamic association constant, independent of ionic interactions, can be calculated which is very close to the calculated thermodynamic association constants for ethidium and proflavine.     

The dynamics of chromatin carcinogen interactions in the human cell.

Human lung epithelioid cells were treated with Benzo (a) pyrene diol epoxide (anti) in order to establish the binding and removal of covalent adducts in chromosomal components. Isolating two different classes of mononucleosomes, it was found that their DNA contained different concentrations of B(a)PDE-DNA adducts, while in both these mononucleosomal preparations only histones H2A and H3 contained detectable amounts of the carcinogen. Further analysis showed that in the intact human cell the carcinogen-DNA adduct distribution is constantly changing as a function of differential accessibility and repair. These results emphasize the dynamics of chromatin-carcinogen modifications.