Binding of enantiomers of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene to polynucleotides

DNA covalent binding studies with enantiomers of trans-7,8-dihydroxy- anti-9,10-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (anti-BPDE) have been carried out by means of spectroscopic techniques (UV, CD, and fluorescence). Synthetic polynucleotides are employed to investigate binding differences between the G.C and A.T base pairs and to elucidate the bases for the stereoselective covalent binding of DNA toward anti-BPDE. The results indicate that of all the polynucleotides studied, only poly(dA-dT).poly(dA-dT) exhibits predominant intercalative covalent binding towards (+)-anti-BPDE and suffers the least covalent modification. Only minor intercalative covalent contributions are found in alternating polymer poly(dA-dC).poly(dG-dT). These observations parallel the DNA physical binding results of anti-BPDE and its hydrolysis products. They support the hypothesis that intercalative covalent adducts derive from intercalative physical binding while the external covalent adducts derive from external bimolecular associations. In contrast to the A.T polymers, the guanine containing polymers exhibit pronounced reduction in covalent modification by (-)-anti-BPDE. The intercalative covalent binding mode becomes relatively more important in the adducts formed by the (-) enantiomer as a consequence of decreased external guanine binding. These findings are consistent with the guanine specificity, stereoselective covalent binding at dG, the absence of stereoselectivity at dA for anti-BPDE, and the enhanced binding heterogeneity for the (-) enantiomer as found in the native DNA studies. The possible sequence and/or conformational dependence of such stereoselective covalent binding is indicated by the opposite pyrenyl CD sign exhibited by (+)-anti-BPDE bound to polynucleotides with pyrimidine on one strand and purine on another vs. that bound to polymers containing alternating purine-pyrimidine sequences.     

Translesion synthesis by human DNA polymerase kappa on a DNA template containing a single stereoisomer of dG-(+)- or dG-(-)-anti-N(2)-BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene)

Several recently discovered human DNA polymerases are associated with translesion synthesis past DNA adducts. These include human DNA polymerase kappa (pol kappa), a homologue of Escherichia coli pol IV, which enhances the frequency of spontaneous mutation. Using a truncated form of pol kappa (pol kappa Delta C), translesion synthesis past dG-(+)- or dG-(-)-anti-N(2)-BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene) adducts was explored. Site-specifically-modified oligodeoxynucleotides containing a single stereoisomeric dG-N(2)-BPDE lesion were used as DNA templates for primer extension reactions catalyzed by pol kappa Delta C. Primer extension was retarded one base prior to the dG-N(2)-BPDE lesion; when incubated for longer times or with higher concentration of enzyme, full primer extension was observed. Quantitative analysis of fully extended products showed preferential incorporation of dCMP, the correct base, opposite all four stereoisomeric dG-N(2)-BPDE lesions. (+)-trans-dG-N(2)-BPDE, a major BPDE-DNA adduct, promoted small amounts of dTMP, dAMP, and dGMP misincorporation opposite the lesion (total 2.7% of the starting primers) and deletions (1.1%). Although (+)-cis-dG-N(2)-BPDE was most effective in blocking translesion synthesis, its miscoding properties were similar to other dG-N(2)-BPDE isomers. Steady-state kinetic data indicate that dCMP is efficiently inserted opposite all dG-N(2)-BPDE adducts and extended past these lesions. The relative frequency of translesion synthesis (F(ins) x F(ext)) of dC.dG-N(2)-BPDE pairs was 2-6 orders of magnitude higher than that of other mismatched pairs. Pol kappa may play an important role in translesion synthesis by incorporating preferentially the correct base opposite dG-N(2)-BPDE. Its relatively low contribution to mutagenicity suggests that other newly discovered DNA polymerase(s) may be involved in mutagenic events attributed to dG-N(2)-BPDE adducts in human cells.     

The reaction of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene with DNA involves attack at the N7-position of guanine moieties

The reaction of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BPDE) with DNA prelabelled with [14C] and [3H]-purine precursors has indicated that in addition to the N2-position of guanine previously reported [10--12] reaction also involves the N7-position of guanine. The hydrocarbon-N7-guanine product was not detected earlier because it is lost from the DNA very readily at pH 7. The same N7-product was obtained by reaction of anti-BPDE with guanine in dimethylformamide.     

Binding of Enantiomers of Trans-7, 8-dihydroxy-anti-9,10-epoxy-7,8,9,10- tetrahydro-benzo [a]pyrene to Polynucleotides

Abstract

DNA covalent binding studies with enantiomers of trans-7,8-dihydroxy- anti-9,10-epoxy- 7,8,9,10-tetrahydro-benzo [a] pyrene (anti-BPDE) have been carried out by means of spectroscopic techniques (UV, CD, and fluorescence). Synthetic polynucleotides are employed to investigate binding differences between the G · C and A · T base pairs and to elucidate the bases for the stereoselective covalent binding of DNA toward anti-BPDE. The results indicate that of all the polynucleotides studied, only poly(dA-dT) · poly(dA-dT) exhibits predominant intercalative covalent binding towards (+)-anti-BPDE and suffers the least covalent modification. Only minor intercalative covalent contributions are found in alternating polymer poly(dA-dC) · poly(dG-dT). These observations parallel the DNA physical binding results of anti-BPDE and its hydrolysis products. They support the hypothesis that intercalative covalent adducts derive from intercalative physical binding while the external covalent adducts derive from external bimolecular associations. In contrast to the A · T polymers, the guanine containing polymers exhibit pronounced reduction in covalent modification by (-)-anti-BPDE. The intercalative covalent binding mode becomes relatively more important in the adducts formed by the (-) enantiomer as a consequence of decreased external guanine binding. These findings are consistent with the guanine specificity, stereoselective covalent binding at dG, the absence of stereoselectivity at dA for anti-BPDE, and the enhanced binding heterogeneity for the (-) enantiomer as found in the native DNA studies. The possible sequence and/or conformational dependence of such stereoselective covalent binding is indicated by the opposite pyrenyl CD sign exhibited by (+)-anti-BPDE bound to polynucleotides with pyrimidine on one strand and purine on another vs. that bound to polymers containing alternating purine-pyrimidine sequences.     

32P-Postlabeling analysis of lipophilic DNA adducts resulting from interaction with (+/-)-3-hydroxy-trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo [a]pyrene.

Bay-region diol epoxides are considered the putative ultimate carcinogens of polynuclear aromatic hydrocarbons. However, the results of studies on tumorigenesis and DNA binding of benzo[a]pyrene (BP) and its bay-region diol epoxide, (+)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyren e [(+)-anti-BPDE] suggest that, in addition to anti-BPDE, other reactive metabolite(s) of BP may also be involved in BP-induced carcinogenesis. Recent studies have demonstrated that 3-hydroxy-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a ]pyrene (anti-BPTE) is another highly reactive metabolite of BP. In order to identify syn- and anti-BPTE-derived DNA adducts and their base selectivity, we synthesized both compounds by two different methods and reacted in vitro with calf thymus DNA and individual nucleotides. The resultant adducts were analyzed by nuclease P1-enhanced 32P-postlabeling. Anti-BPTE produced three major and several minor adducts with DNA; dAp and dGp were the preferred substrates, while dCp and dTp were the least reactive. In contrast, syn-BPTE produced two major adducts each with DNA and dGp; dAp generated only one adduct. Co-chromatography of anti-BPTE-derived DNA adducts with those of mononucleotide adducts revealed that the major adducts in DNA were guanine derived. Further, co-chromatographic results revealed that the anti-BPTE-DNA adducts were distinctly different from that of anti-BPDE-DNA adducts. These observations indicate that both syn- and anti-BPTE can react with DNA bases and these DNA adducts may also contribute to BP-induced carcinogenesis.     

Error-prone translesion synthesis by human DNA polymerase eta on DNA-containing deoxyadenosine adducts of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

When human DNA polymerase eta (pol eta) encounters N6-deoxyadenosine adducts formed by trans epoxide ring opening of the 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP DE) isomer with (+)-7R,8S,9S,10R configuration ((+)-BaP DE-2), misincorporation of A or G and incorporation of the correct T are equally likely to occur. On the other hand, the enzyme exhibits a 3-fold preference for correct T incorporation opposite adducts formed by trans ring opening of the (-)-(7S,8R,9R,10S)-DE-2 enantiomer. Adducts at dA formed by cis ring opening of these two BaP DE-2 isomers exhibit a 2-3-fold preference for A over T incorporation, with G intermediate between the two. Extension one nucleotide beyond these adducts is generally weaker than incorporation across from them, but among mismatches the (adducted A*) x A mispair is the most favored for extension. Because mutations can only occur if mispairs are extended, this observation is consistent with the occurrence of A x T to T x A transversions as common mutations in animal cells treated with BaP DE-2 isomers. Adducts with S absolute configuration at the point of attachment of the hydrocarbon to the base inhibit incorporation and extension by pol eta to a lesser extent than their R counterparts. Template-primers containing each of the four isomeric dA adducts derived from BaP DE-2 and two adducts derived from 9,10-epoxy-7,8,9,10-tetrahydrobenzo-[a]pyrene in which the 7- and 8-hydroxyl groups of the DEs are replaced with hydrogens exhibit reduced electrophoretic mobilities relative to the unadducted oligonucleotides. This effect is largely independent of DNA sequence. Decreased mobility correlates with an increased rate of incorporation by pol eta, suggesting a systematic relationship between the overall DNA structure and efficiency of the enzyme.     

DNA repair and replication in human fibroblasts treated with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

DNA repair and replication were examined in diploid human fibroblasts after treatment with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I). Unscheduled DNA synthesis exhibited a linear response to BPDE-I concentrations up to 1.5 microM and a saturation plateau after higher concentrations. Maximal unscheduled DNA synthesis was observed in the first hour after treatment with synthesis diminishing progressively thereafter. Half-maximal unscheduled DNA synthesis was seen within 4-6 h after treatment with 0.7 microM BPDE-I. DNA replication was inhibited by BPDE-I in a dose- and time-dependent fashion. The mechanisms of this inhibition were characterized by velocity sedimentation of pulse-labeled nascent DNA in alkaline sucrose gradients. Very low concentrations of BPDE-I (0.03 and 0.07 microM) were found to inhibit replicon initiation by up to 50% within 30-60 min after treatment. Recovery of initiation following these low concentrations was evident within 3 h after treatment. Higher concentrations of carcinogen inhibited DNA synthesis in active replicons. This effect was manifested by a reduction in incorporation of precursor into replication intermediates of greater than 1 X 10(7) Da with the concurrent production of abnormally small nascent DNA. When viewed 45 min after treatment with 0.17 microM BPDE-I the combination of these two effects partially masked the inhibition of replicon initiation. However, even after treatment with 0.33 microM BPDE-I an effect on initiation was evident. These results reveal a pattern of response to BPDE-I that is quite similar to that produced by 254 nm radiation.     

DNA repair and replication in human fibroblasts treated with (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

DNA repair and replication were examined in diploid human fibroblasts after treatment with (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I). Unscheduled DNA synthesis exhibited a linear response to BPDE-I concentrations up to 1.5 μM and a saturation plateau after higher concentrations. Maximal unscheduled DNA synthesis was observed in the first hour after treatment with synthesis diminishing progressively thereafter. Half-maximal unscheduled DNA synthesis was seen within 4–6 h after treatment with 0.7 μM BPDE-I. DNA replication was inhibited by BPDE-I in a dose- and time-dependent fashion. The mechanisms of this inhibition were characterized by velocity sedimentation of pulse-labeled nascent DNA in alkaline sucrose gradients. Very low concentrations of BPDE-I (0.03 and 0.07 μM) were found to inhibit replicon initiation by up to 50% within 30–60 min after treatment. Recovery of initiation following these low concentrations was evident within 3 h after treatment. Higher concentrations of carcinogen inhibited DNA synthesis in active replicons. This effect was manifested by a reduction in incorporation of precursor into replication intermediates of greater than 1·10⁷ Da with the concurrent production of abnormally small nascent DNA. When viewed 45 min after treatment with 0.17 μM BPDE-I the combination of these two effects partially masked the inhibition of replicon initiation. However, even after treatment with 0.33 μM BPDE-I an effect on initiation was evident. These results reveal a pattern of response to BPDE-I that is quite similar to that produced by 254 nm radiation.     

Solution structure of a cis-opened (10R)-N6-deoxyadenosine adduct of (9S,10R)-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in a DNA duplex

The solution structure of an 11-mer DNA duplex, d(CGGTCA*CGAGG) x d(CCTCGTGACCG), containing a 10R adduct at dA* that corresponds to the cis addition of the N(6)-amino group of dA(6) to (+)-(9S,10R)-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene was studied by 2D NMR methods. The NOESY cross-peak patterns indicate that the hydrocarbon is intercalated on the 5'-side of the modified base. This observation is the same as that observed for other oligonucleotides containing (10R)-dA adducts but opposite to that observed for the corresponding (10S)-dA adducts which are intercalated on the 3'-side of the modified base. The hydrocarbon is intercalated from the major groove without significant disruption of either the anti glycosidic torsion angle of the modified residue or the base pairing of the modified residue with the complementary residue on the opposite strand. The ensemble of 10 structures determined exhibits relatively small variations (6-15 degrees) in the characteristic hydrocarbon-base dihedral angles (alpha' and beta') as well as the glycosidic torsion angle chi. These angles are similar to those in a previously determined cis-opened benzo[a]pyrene diol epoxide-(10R)-dA adduct structure. Comparison of the present structure with the cis-opened diol epoxide adduct suggests that the absence of the 7- and 8-hydroxyl groups results in more efficient stacking of the aromatic moiety with the flanking base pairs and deeper insertion of the hydrocarbon into the helix. Relative to normal B-DNA, the duplex containing the present tetrahydroepoxide adduct is unwound at the lesion site, whereas the diol epoxide adduct structure is more tightly wound than normal B-DNA. Buckling of the adducted base pair as well as the C(5)-G(18) base pair that lies immediately above the hydrocarbon is much less severe in the present adducted structure than its cis-opened diol epoxide counterpart.     

Solution structure of a trans-opened (10S)-dA adduct of (+)-(7S,8R,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in a fully complementary DNA duplex: evidence for a major syn conformation

Two-dimensional NMR was used to determine the solution structure of an undecanucleotide duplex, d(CGGTCACGAGG).d(CCTCGTGACCG), in which (+)-(7S,8R,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene is covalently bonded to the exocyclic N(6)() amino group of the central deoxyadenosine, dA(6), through trans addition at C10 of the epoxide (to give a 10S adduct). The present study represents the first NMR structure of a benzo[a]pyrene (10S)-dA adduct in DNA with a complementary T opposite the modified dA. Exchangeable and nonexchangeable protons of the modified duplex were assigned by the use of TOCSY (in D(2)O) and NOESY spectra (in H(2)O and D(2)O). Sequential NOEs expected for a B-type DNA conformation with typical Watson-Crick base pairing are observed along the duplex, except at the lesion site. We observed a strong intraresidue NOE cross-peak between H1' and H8 of the modified dA(6). The sugar H2' and H2' ' of dC(5) lacked NOE cross-peaks with H8 of dA(6) but showed weak interactions with H2 of dA(6) instead. In addition, the chemical shift of the H8 proton (7.51 ppm) of dA(6) appears at a higher field than that of H2 (8.48 ppm). These NOE and chemical shift data for the dA(6) base protons are typical of a syn glycosidic bond at the modified base. Restrained molecular dynamics/energy minimization calculations show that the hydrocarbon is intercalated from the major groove on the 3'-side of the modified base between base pairs A(6)-T(17) and C(7)-G(16) and confirm the syn glycosidic angle (58 degrees ) of the modified dA(6). In the syn structure, a weak A-T hydrogen bond is possible between the N3-H proton of T(17) and N7 of dA(6) (at a distance of 3.11 A), whereas N1, the usual hydrogen bonding partner for N3-H of T when dA is in the anti conformation, is 6.31 A away from this proton. The 10(S)-dA modified DNA duplex remains in a right-handed helix, which bends in the direction of the aliphatic ring of BaP at about 42 degrees from the helical axis. ROESY experiments provided evidence for interconversion between the major, syn conformer and a minor, possibly anti, conformer.     

High-density lipoproteins decrease both DNA binding and mutagenicity of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in V79 Chinese hamster cells

The effects of separate lipoproteins or of serum with high or low lipoprotein concentrations on formation of lipophilic carcinogen adducts with DNA and on mutagenicity of the carcinogen was investigated using V79 Chinese hamster lung cells. Binding of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) to DNA and BPDE induction of 6-thioguanine (6-TG)-resistant mutants in V79 cells was significantly lower after 1 or 4 h when the medium was supplemented with purified HDL, and was lower after 1 h but not 4 h when the medium was supplemented with serum containing a high concentration of mixed lipoproteins (LP). Cells grown in medium without serum or LP supplementation exhibited the highest levels of both BPDE-DNA adduct formation and mutagenesis after 1 h. At 1 h, cells exposed to BPDE in LDL-supplemented medium showed decreased adduct formation and mutagenesis when compared to cells treated with BPDE in PBS-supplemented medium. After 4 h, cells treated with BPDE in LDL-supplemented medium gave the highest levels of adduct formation and the highest mutation frequency. These results suggest that both LDL and HDL effectively decrease the concentration of BPDE available to V79 cells exposed to the mutagen for short periods of time, resulting in decreased interaction of BPDE with DNA and decreased BPDE-associated mutagenesis, but that both BPDE-DNA adduct formation and mutagenesis increased as a function of increased exposure time in the presence of LDL. The results suggest that LDL, but not HDL, uptake by adsorptive endocytosis may be associated with potentiated entry of BPDE into V79 cells as a function of time.     

Human lymphocytes treated with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene require low-density lipoproteins for DNA excision repair

Human lymphocytes which were non-mitogen-stimulated, and which were depleted of lipoproteins, were found to be deficient in DNA excision repair typically initiated in these cells in response to treatment with a direct-acting polynuclear aromatic hydrocarbon carcinogen. Lymphocytes either depleted of lipoproteins or supplemented with human low-density lipoproteins formed DNA-carcinogen adducts which were not chromatographically distinguishable. The state of lipoprotein depletion did not alter lymphocyte uptake of thymidine from the medium. Lymphocytes which were depleted of lipoproteins, treated with carcinogen, and subsequently supplemented with low-density lipoproteins, regained the ability to engage in DNA excision repair. The data suggest that either low-density lipoprotein(s), or a component(s) of low-density lipoprotein(s), is required by human lymphocytes in order to initiate excision repair of carcinogen-damaged DNA.     

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.     

MINDO/3 calculations of the conformation and carcinogenicity of epoxy-metabolites of aromatic hydrocarbons: 7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene

Quantum mechanical calculations in the MINDO/3 approximation were performed on the four conformations of the alicyclic moiety of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene.Total charge and frontier orbital densities show that attack by nucleophiles will occur predominantly at Position 10 of all configurations of the dihydroxyepoxybenzo(a)pyrene.The calculations show the cis diastereomer to be more stable than the trans, although no evidence for hydrogen bonding in the (ax, ax′) conformer was found.On the basis of the results obtained for the stability of the various conformers, a model is proposed to explain the higher carcinogenicity of the trans isomer as compared to the cis. Such a model implies the formation of an intercalation complex between the diol epoxide metabolite and nucleic acids.     

Amino acid substitutions at positions 207 and 221 contribute to catalytic differences between murine glutathione S-transferase Al-1 and A2-2 toward (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene.

We have previously identified a novel Alpha class murine glutathione (GSH) S-transferase isoenzyme (designated mGSTAl-2) which is exceptionally efficient in catalyzing the GSH conjugation of (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], the ultimate carcinogen of widespread environmental pollutant benzo[a]pyrene. Furthermore, we have demonstrated that the Al-type subunit of this isoenzyme is significantly more active toward (+)-anti-BPDE than the other subunit (mGSTA2). To establish the basis for catalytic differences between mGSTAl and mGSTA2, which differ in their primary structures by 10 amino acids [distributed in three sections (I-III) as clusters of two (residues 65 and 95), three (residues 157, 162, and 169), and five (residues 207, 213, 218, 221, and 222) amino acids], three chimeric enzymes were expressed and tested for their activity toward (+)-anti-BPDE. These studies revealed that amino acid substitution(s) in section III determined the high catalytic activity of mGSTAl. Molecular modeling studies suggested that amino acid substitutions at positions 207 and/or 221, but not at positions 213, 218, and 222, may be responsible for such a difference. To test this possibility, amino acids at positions 207 and 221 of mGSTAl were mutated with the equivalent residues of mGSTA2. Kinetic analysis of the wild type and the mutant enzymes revealed that both methionine-207 and isoleucine-221 are critical for higher activity of mGSTA1-1 toward (+)-anti-BPDE compared with that of mGSTA2-2.     

Gram-scale synthesis of (+)- and (-)-trans-10-amino-9-hydroxy-9,10-dihydrophenanthrene by enzymatic hydrolysis

The enzymatic resolution of (+/-)-trans-10-azido-9-acetoxy-9,10-dihydrophenanthrene by Candida cylindracea lipase-catalyzed hydrolysis was achieved on the gram-scale with excellent yield and enantiomeric excess. The resulting (+)- and (-)-amino alcohols were derivatized to alpha-hydroxy-N-benzenesulphonamides.     

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).     

Covalent binding of photosensitive 1-(12-azido-[9,10(-3)H2]oleoyl)glycero-3-phosphocholine (lysolecithin) to human serum high density apolipoproteins.

Human serum high density apoproteins were complexed with increasing concentrations of 1-(12-azido-[9,10(-3)H2]oleoyl)glycero-3-phosphocholine up to the saturation concentration (72 mol lysolecithin per mol apo HDL). Ultraviolet irradiation generated the nitrene which led to crosslinking with the two main apolipoproteins AI and AII. Methods are described for the removal of excess, unbound lipid and the column chromatographic separation of the lipopolypeptides AI and AII.     

Facilitation of the conversion from B to Z conformation in poly(dG-dC) modified by anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide

The transition from B to Z conformation has been studied in poly(dG-dC) covalently modified with racemic anti- or syn-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), a strong and a weak carcinogen, respectively. Circular dichroism was used to study the kinetics of the transition after a sudden increase of the ionic strength to 2.7 M NaCl. The results show that the rate of the B to Z transition of poly(dG-dC) in high NaCl concentration is considerably enhanced by bound anti-BPDE and diminished by bound syn-BPDE. The results may be interpreted such that at the binding site of anti-BPDE the base stacking is distorted and made looser, which facilitates the B to Z transition. The partly intercalative nature of the syn-BPDE complexes apparently is effective in reducing the rate of the transition. These properties of the two BPDEs may be relevant to explain their different carcinogenic potencies.