Preferential misincorporation of purine nucleotides by human DNA polymerase eta opposite benzo[a]pyrene 7,8-diol 9,10-epoxide deoxyguanosine adducts

Human DNA polymerase eta was used to copy four stereoisomeric deoxyguanosine (dG) adducts derived from benzo[a]pyrene 7,8-diol 9,10-epoxide (diastereomer with the 7-hydroxyl group and epoxide oxygen trans (BaP DE-2)). The adducts, formed by either cis or trans epoxide ring opening of each enantiomer of BaP DE-2 by N(2) of dG, were placed at the fourth nucleotide from the 5'-end in two 16-mer sequence contexts, 5' approximately CG*A approximately and 5' approximately GG*T. poleta was remarkably error prone at all four diol epoxide adducts, preferring to misincorporate G and A at frequencies 3- to more than 50-fold greater than the frequencies for T or the correct C, although the highest rates were 60-fold below the rate of incorporation of C opposite a non-adducted G. Anti to syn rotation of the adducted base, consistent with previous NMR data for a BaP DE-2 dG adduct placed just beyond a primer terminus, provides a rationale for preferring purine misincorporation. Extension of purine misincorporations occurred preferentially, but extension beyond the adduct site was weak with V(max)/K(m) values generally 10-fold less than for misincorporation. Mostly A was incorporated opposite (+)-BaP DE-2 dG adducts, which correlates with published observations that G --> T is the most common type of mutation that (+)-BaP DE-2 induces in mammalian cells.     

A benzo[a]pyrene -7,8-dihydrodiol-9,10-epoxide is the major metabolite involved in the binding of benzo[a]pyrene to DNA in isolated viable rat hepatocytes

Benzo[a]pyrene is metabolised by isolated viable hepatocytes from both untreated and 3-methylcholanthrene pretreated rats to reactive metabolites which covalently bind to DNA. The DNA from the hepatocytes was isolated, purified and enzymically hydrolysed to deoxyribonucleosides. The hydrocarbon-deoxyribonucleoside products after initial separation, on small columns of Sephadex LH-20, from unhydrolysed DNA, oligonucleotides and free bases, were resolved by high pressure liquid chromatography (HPLC). The qualitative nature of the adducts found in both control and pretreated cells was virtually identical; however pretreatment with 3-methylcholanthrene resulted in a quantitatively higher level of binding. The major hydrocarbon-deoxyribonucleoside adduct, found in hepatocytes co-chromatographed with that obtained following reaction of the diol-epoxide, (±)7α,8β-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene with DNA. Small amounts of other adducts were also present including a more polar product which co-chromatographed with the major hydrocarbon-deoxyribonucleoside adduct formed following microsomal activation of 9-hydroxybenzo[a]pyrene and subsequent binding to DNA. In contrast to the results with hepatocytes, when microsomes were used to metabolically activate benzo[a]pyrene, the major DNA bound-product co-chromatographed with the more polar adduct formed upon further metabolism of 9-hydroxybenzo[a]pyrene. These results illustrate that great caution must be exercised in the extrapolation of results obtained from short-term mutagenesis test systems, utilising microsomes, to in vivo carcinogenicity studies.     

Efficiency and accuracy of SOS-induced DNA polymerases replicating benzo[a]pyrene-7,8-diol 9,10-epoxide A and G adducts.

Nucleotide incorporation fidelity, mismatch extension, and translesion DNA synthesis efficiencies were determined using SOS-induced Escherichia coli DNA polymerases (pol) II, IV, and V to copy 10R and 10S isomers of trans-opened benzo[a]pyrene-7,8-diol 9,10-epoxide (BaP DE) A and G adducts. A-BaP DE adducts were bypassed by pol V with moderate accuracy and considerably higher efficiency than by pol II or IV. Error-prone pol V copied G-BaP DE-adducted DNA poorly, forming A*G-BaP DE-S and -R mismatches over C*G-BaP DE-S and -R correct matches by factors of approximately 350- and 130-fold, respectively, even favoring G*G-BaP DE mismatches over correct matches by factors of 2-4-fold. In contrast, pol IV bypassed G-BaP DE adducts with the highest efficiency and fidelity, making misincorporations with a frequency of 10(-2) to 10(-4) depending on sequence context. G-BaP DE-S-adducted M13 DNA yielded 4-fold fewer plaques when transfected into SOS-induced DeltadinB (pol IV-deficient) mutant cells compared with the isogenic wild-type E. coli strain, consistent with the in vitro data showing that pol IV was most effective by far at copying the G-BaP DE-S adduct. SOS polymerases are adept at copying a variety of lesions, but the relative contribution of each SOS polymerase to copying damaged DNA appears to be determined by the lesion's identity.     

Effects of pH and salt concentration on the hydrolysis of a benzo[alpha]pyrene 7,8-diol-9,10-epoxide catalyzed by DNA and polyadenylic acid

The time-dependent absorbance change that occurs when benzo[alpha]pyrene 7,8-diol-9,10-epoxide is added to solutions of calf thymus DNA has been shown, by an unequivocal chromatographic method, to correspond to DNA-catalyzed hydrolysis of the diol-epoxide. At 25 degrees C and mu = 0.10, the kinetics of the reaction of the diol-epoxide with polyadenylic acid or DNA are consistent with preequilibrium formation of a non-covalent complex between the diol-epoxide and the polynucleotide or DNA, followed by hydrolysis of the bound epoxide by a process that is first-order in hydronium ions. Cacodylic acid also catalyzes the hydrolysis of the epoxide bound to polyadenylic acid. The rate of the DNA-catalyzed hydrolysis exhibits little or no enantiomeric selectivity for the diol-epoxide. DNA catalyzed hydrolysis of the diol-epoxide is extraordinarily sensitive to the salt concentration in the reaction medium: the rate of hydrolysis of the bound epoxide at pH 7 is retarded by a factor of approximately 45 in the presence of 0.1 M sodium chloride compared to a 1 mM buffer containing no added salt. Thus, studies of the interactions of DNA with carcinogenic diol-epoxides must take into account the ionic environment of DNA within the cell.     

Modulation of the cytotoxicity and mutagenicity of benzo[a]pyrene and benzo[a]pyrene 7,8-diol by glutathione and glutathione S-transferases in mammalian cells (CHO/HGPRT assay)

Biologically reactive metabolites of benzo[a]pyrene (BP) and benzo[a]-pyrene 7,8-diol (BP-diol), formed by the mixed-function oxidase (MFO) system, are substrates for conjugation and detoxication by glutathione (GSH) when catalyzed by glutathione S-transferases (GSHT). We have investigated the detoxication of BP- and BP-diol-induced cytotoxicity and mutagenicity with GSH by supplementing the S9 mix used in the Chinese hamster ovary cells/hypoxanthine-guanine phosphoribosyltransferase (CHO/HGPRT) assay with GSH (6.5 mM) or GSH plus GSHT. The addition of GSH to the S9 mix resulted in a reduction of BP- and BP-diol induced cytotoxicity. GSH plus GSHT eliminated BP-induced cytotoxicity and reduced the mutagenicity of BP. GSH inhibited the mutagenicity at low (essentially non-lethal) concentrations of BP-diol, but did not do so at toxic concentrations. GSH plus GSHT inhibited the cytotoxicity and mutagenicity of BP-diol at concentrations not affected by GSH alone. These studies indicate that biochemical mechanisms of detoxication can affect the biological activity of a carcinogen, such as BP or BP-diol as profoundly as bioactivation by the MFO system.     

Ellagic acid toxicity and interaction with benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol in human bronchial epithelial cells

Ellagic acid, a plant phenol present in various foods consumed by humans, has been reported to have both anti-mutagenic and anti-carcinogenic potential. To evaluate the potential anti-carcinogenic property of ellagic acid, we tested its effects on the toxicity of ben-zo[a]pyrene and benzo[a]pyrene, 7,8-dihydrodiol and binding of benzo[a]yrene to DNA in cultured human bronchial epithelial cells. The toxicity of ellagic acid itself for human bronchial epithelial cells was also determined. Using a colony-forming efficiency assay, it was found that a nontoxic concentration of ellagic acid (5 g/ml) enhanced the toxicity of benzo[a]pyrene.7,8-dihydrodiol in human bronchial epithelial cells. In contrast, ellagic acid at concentrations of l.5 and 3.0 g/ml inhibited binding of benzo[a]pyrenemetabolites to DNA in these cells. An explanation for the potentiating effect of ellagic acid on the toxicity of benzo[a]pyrene, 7,8-dihydrodiol will require further investigation into the possible mechanisms of interaction between these two compounds.Abbreviations B[a]P benzo[a]pyrene - B[a]P 7,8-DHD (±)trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene - B[a]PDE-1 (±)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - B[a]PDE-2 (±) 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - B[a]PDE-1:dG N²-]10{7,8,9-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl}:deoxyguanosine - B[a]PDE-2:dG NZ-{10-[7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl}:deoxyguanosine - CFE colony forming efficiency - EA ellagic acid - HBE human bronchial epithelial     

Structural basis for catalytic differences between alpha class human glutathione transferases hGSTA1-1 and hGSTA2-2 for glutathione conjugation of environmental carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide

The ultimate diol epoxide carcinogens derived from polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (BP), are metabolized primarily by glutathione (GSH) conjugation reaction catalyzed by GSH transferases (GSTs). In human liver and probably lung, the alpha class GSTs are likely to be responsible for the majority of this reaction because of their high abundance. The catalytic efficiency for GSH conjugation of the carcinogenic (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide [(+)-anti-BPDE] is more than 5-fold higher for hGSTA1-1 than for hGSTA2-2. Here, we demonstrate that mutation of isoleucine-11 of hGSTA2-2, a residue located in the hydrophobic substrate-binding site (H-site) of the enzyme, to alanine (which is present in the same position in hGSTA1-1) results in about a 7-fold increase in catalytic efficiency for (+)-anti-BPDE-GSH conjugation. Thus, a single amino acid substitution is sufficient to convert hGSTA2-2 to a protein that matches hGSTA1-1 in its catalytic efficiency. The increased catalytic efficiency of hGSTA2/I11A is accompanied by greater enantioselectivity for the carcinogenic (+)-anti-BPDE over (-)-anti-BPDE. Further remodeling of the H-site of hGSTA2-2 to resemble that of hGSTA1-1 (S9F, I11A, F110V, and S215A mutations, SIFS mutant) results in an enzyme whose catalytic efficiency is approximately 13.5-fold higher than that of the wild-type hGSTA2-2, and about 2.5-fold higher than that of the wild-type hGSTA1-1. The increased activity upon mutations can be rationalized by the interactions of the amino acid side chains with the substrate and the orientation of the substrate in the active site, as visualized by molecular modeling. Interestingly, the catalytic efficiency of hGSTA2-2 toward (-)-anti-BPDE was increased to a level close to that of hGSTA1-1 upon F110V, not I11A, mutation. Similar to (+)-anti-BPDE, however, the SIFS mutant was the most efficient enzyme for GSH conjugation of (-)-anti-BPDE.     

A single site-specific trans-opened 7,8,9,10-tetrahydrobenzo[a]pyrene 7,8-diol 9,10-epoxide N2-deoxyguanosine adduct induces mutations at multiple sites in DNA

Site-specific mutagenicity of trans-opened adducts at the exocyclic N(2)-amino group of guanine by the (+)-(7R,8S,9S,10R)- and (-)-(7S,8R,9R,10S)-enantiomers of a benzo[a]pyrene 7,8-diol 9,10-epoxide (7-hydroxyl and epoxide oxygen are trans, BPDE-2) has been determined in Chinese hamster V79 cells and their repair-deficient counterpart, V-H1 cells. Four vectors containing single 10S-BPDE-dG or 10R-BPDE-dG adducts positioned at G(0) or G(-1) in the analyzed 5'-ACTG(0)G(-1)GA sequence of the non-transcribed strand were separately transfected into the cells. Mutations at each of the seven nucleotides were analyzed by a novel primer extension assay using a mixture of one dNTP complementary to the mutated nucleotide and three other ddNTPs and were optimized to quantify levels of a mutation as low as 1%. Only G --> T mutations were detected at the adducted sites; the 10S adduct derived from the highly carcinogenic (+)-diol epoxide was 40-50 and 75-140% more mutagenic than the 10R adduct in V79 and V-H1 cells, respectively. Importantly, the 10S adducts, but not the 10R adducts, induced separate non-targeted mutations at sites 5' to the G(-1) and G(0) lesions (G(0) --> T and C --> T, respectively) in both cell lines. Neither the T 5' to G(0) nor sites 3' to the lesions showed mutations. Non-targeted mutations may enhance overall mutagenicity of the 10S-BPDE-dG lesion and contribute to the much higher carcinogenicity and mutagenicity of (+)-BPDE-2 compared with its (-)-enantiomer. Our study reports a definitive demonstration of mutations distal to a site-specific polycyclic aromatic hydrocarbon adduct.     

Metabolism of benzo[a]pyrene VI. Stereoselective metabolism of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol epoxides

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (?)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (?)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (?)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (?)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ～30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ～5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.     

Glutathione S-transferase-catalyzed conjugation of 9,10-epoxystearic acid with glutathione

The possible role of glutathione S-transferases (GST) in detoxification of fatty acid epoxides generated during lipid peroxidation has been evaluated. Present studies showed that cytosolic human glutathione S-transferases belonging to alpha, mu, and pi classes isolated from human liver and lung catalyzed the conjugation of glutathione and 9,10-epoxystearic acid. The product of enzymatic reaction, i.e., conjugate of GSH and epoxystearic acid, was isolated and characterized. The Michaelis constant (Km) values of the alpha, mu, and pi classes of GSTs for 9,10-epoxystearic acid were found to be 0.47, 0.32 and 0.80 mM, respectively, whereas the maximal velocity (V max) values for the alpha, mu, and pi classes of GSTs were found to be 142, 256, and 52 mol/min/mol, respectively. These results indicate that even though 9,10-epoxystearic acid is a substrate for all the three classes of GSTs, the mu class isozymes have maximum activity toward this substrate and may preferentially metabolize fatty acid epoxides more effectively as compared to the other classes of GSTs.     

Status of reduced glutathione in primary cultures of rat hepatocytes and the effect on conjugation of benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide

The intracellular level of reduced glutathione (GSH) and GSH conjugation have been investigated in primary cell cultures of hepatocytes isolated from control rats, phenobarbitone (PB) and 3-methylcholanthrene (MC) treated rats. The data demonstrate that in all cell cultures the GSH concentrations show a triphasic pattern: (i) within 1 h of culture an initial marked decrease to 50% of the levels found in fresh hepatocytes; (ii) recovery of GSH concentrations to above the levels observed in fresh cells. This occurs after 6 h in culture with control cells and after 10-24 h with cells from either PB or MC treated rats and was most prominent in cells from PB-treated rats. (iii) A slow decline to between 30 and 40 nmol GSH/mg protein from 24 to 96 h in culture. Synthesis of GSH was slower in cultured cells from PB treated rats and this was confirmed by the resynthesis rates when diethylmaleate (DEM) was used to deplete GSH. The formation of GSH conjugates with racemic 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) was measured in control cells in suspension and after 3 and 24 h in culture. Despite the decrease in GSH concentrations observed between 1 and 4 h after culture, the conjugation rates were not decreased.     

Metabolism of benzo[a]pyrene in human skin xenografts.

Although a human neonatal foreskin graft to a nude mouse has been shown to be morphologically intact for several months after establishment, the feasibility of using this system for carcinogenesis studies has not been widely investigated. In this study, we have investigated the metabolism of benzo[a]pyrene (BaP) in human skin xenografts after the topical application of different concentrations of [3H]BaP (0.5 microgram-10.0 micrograms and 20 muCi/graft) for 2 h and 1.0 micrograms [3H]BaP for various intervals of time up to 4 h. Significant amounts of different organic solvent soluble metabolites were observed in all the different samples. The increase in the amounts of the organic solvent soluble metabolites was linear over the 0.5 microgram to 5.0 micrograms/graft range. When 1.0 microgram [3H]BaP was applied to each graft, the maximum production of the organic solvent soluble metabolites was observed 10 minutes after treatment and it then decreased with time. Diols were the major metabolites detected in each of the experiments, followed by phenols, and then tetrols. The levels of water soluble glucuronide and sulfate conjugates were almost equivalent for each treatment over the same range of application of BaP per graft. The combined levels of these conjugates and the non-extractable organic soluble metabolites in the residue, generally ranged between 7-24% of the total metabolites in the various experiments.     

Benzo[a]pyrene-7,8-diol-9,10-epoxide causes caspase-mediated apoptosis in H460 human lung cancer cell line

We have shown previously that wild-type p53 renders H460 human lung cancer cells more sensitive to apoptosis induction by environmental carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), but the mechanism of cell death is not fully understood. The present study provides insights into the mechanism by which BPDE causes apoptosis in H460 cells. Exposure of H460 cells to BPDE resulted in a concentration-dependent apoptotic cell death characterized by cleavage of poly(ADP-ribose)polymerase, DNA condensation, and apoptotic histone-associated DNA fragments released into the cytosol. The BPDE-mediated release of apoptotic histone-associated DNA fragments into the cytosol was also observed in a normal bronchial epithelial cell line BEAS-2B. The BPDE-induced apoptosis in H460 cells correlated with up-regulation of pro-apoptotic protein Bak, down-regulation of anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xL, release of cytochrome c from mitochondria to the cytosol without a change in mitochondrial membrane potential or mitochondrial morphology (electron microscopy), and cleavage of caspase-8, -9, and -3. Ectopic expression of Bcl-2 failed to confer significant protection against BPDE-induced apoptosis in H460 cells. The SV40 immortalized mouse embryonic fibroblasts (MEFs) derived from Bak and Bax double knockout mice, but not Bid knockout mice, were significantly more resistant to BPDE-induced apoptosis compared with the MEFs derived from wild-type mice. The BPDE-induced apoptosis was partially but statistically significantly attenuated in the presence of specific inhibitors of caspase-9 (z-LEHDfmk) and caspase-8 (z-IETDfmk). In conclusion, the present study reveals that BPDE-induced apoptosis in H460 cells is associated with Bak induction and caspase activation but independent of Bcl-2.     

The role of the Ah receptor and p38 in benzo[a]pyrene-7,8-dihydrodiol and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide-induced apoptosis

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants in the environment. Benzo[a]pyrene (B[a]P), a prototypical member of this class of chemicals, affects cellular signal transduction pathways and induces apoptosis. In this study, the proximate carcinogen of B[a]P metabolism, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-dihydrodiol) and the ultimate carcinogen, B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide(+/-) (BPDE-2) were found to induce apoptosis in human HepG2 cells. Apoptosis initiated by B[a]P-7,8-dihydrodiol was linked to activation of the Ah receptor and induction of CYP1A1, an event that can lead to the formation of BPDE-2. With both B[a]P-7,8-dihydrodiol and BPDE-2 treatment, changes in anti- and pro-apoptotic events in the Bcl-2 family of proteins correlated with the release of mitochondrial cytochrome c and caspase activation. The onset of apoptosis as monitored by caspase activation was linked to mitogen-activated protein (MAP) kinases. Utilizing mouse hepa1c1c7 cells and the Arnt-deficient BPRc1 cells, activation of MAP kinase p38 by B[a]P-7,8-dihydrodiol was shown to be Ah receptor-dependent, indicating that metabolic activation by CYP1A1 was required. This was in contrast to p38 activation by BPDE-2, an event that was independent of Ah receptor function. Confirmation that MAP kinases play a critical role in BPDE-2-induced apoptosis was shown by inhibiting caspase activation of poly(ADP-ribose)polymerase 1 (PARP-1) by chemical inhibitors of p38 and ERK1/2. Furthermore, mouse embryo p38-/- fibroblasts were shown to be resistant to the actions of BPDE-2-induced apoptosis as determined by annexin V analysis, cytochrome c release, and cleavage of PARP-1. These results confirm that the Ah receptor plays a critical role in B[a]P-7,8-dihydrodiol-induced apoptosis while p38 MAP kinase links the actions of an electrophilic metabolite like BPDE-2 to the regulation of programmed cell death.     

Sulfate conjugation of benzo[alpha]pyrene metabolites and derivates

Sulfate conjugation of benzo[alpha]pyrene(BP) metabolites and derivatives was studied. The reaction sequence consisted of two steps; activation of sulfate ion to 3'-phosphoadenosine-5'-phosphosulfate and transfer of the activated sulfate to the BP-derivatives. Both reactions were carried out by enzymes located in the rat liver 105 000 g supernatant. The reactions required MgCl2. Phenol and quinone derivatives were generally good substrates for sulfate conjugation and different reactivities were observed with the dihydrodiol derivatives. Sulfate conjugates were more polar than their parent BP-derivatives and except for quinone conjugates were easily extracted with ethyl acetate. The role of sulfate conjugation in BP carcinogenesis is discussed.     

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.     

Epoxy derivatives of aromatic polycyclic hydrocarbons. The preparation and metabolism of epoxides related to benzo[a]pyrene and to 7,8- and 9,10-dihydrobenzo[a]pyrene

Products that appeared to be mainly benzo[a]pyrene 7,8-oxide and benzo[a]pyrene 9,10-oxide were synthesized and their chemical and biochemical properties were investigated. The oxides were unstable and readily rearranged to phenols. They were converted by rat liver homogenates and microsomal preparations into phenols and dihydrodiols, but glutathione conjugates were not formed in appreciable amounts. The dihydrodiols formed from benzo[a]pyrene 7,8- and 9,10-oxide by rat liver microsomal preparations were identical in their chromatographic and spectrographic properties with dihydrodiols formed when benzo[a]pyrene was metabolized by rat liver homogenates. 9,10-Dihydrobenzo[a]pyrene 7,8-oxide and 7,8-dihydrobenzo[a]pyrene 9,10-oxide were also synthesized. They were converted by rat liver homogenates and microsomal preparations into the related cis- and trans-dihydroxy compounds. Glutathione conjugates were formed from the oxides by rat liver homogenates. Both 7,8- and 9,10-dihydrobenzo[a]pyrene were metabolized by rat liver homogenates to mainly the trans-isomers of the related dihydroxy compounds. In experiments with boiled homogenates, the benzo[a]pyrene oxides were converted into phenols, whereas the dihydrobenzo[a]pyrene oxides yielded small amounts of the related dihydroxy compounds.