Jan Szeliga

Application of mixed mobile phases and a step gradient method in capillary electrochromatography for the separation of isomeric polycyclic aromatic hydrocarbon-deoxyribonucleoside adduct mixtures prepared in vitro

Capillary electrochromatography (CEC) was used for the analysis of mixtures of neutral isomeric compounds derived from the reaction of carcinogenic hydrocarbon (benzo[g]chrysene and 5,6-dimethylchrysene) dihydrodiol epoxides with calf thymus deoxyribonucleic acid (DNA). The CEC analysis demonstrated higher resolution, greater speed and lower analyte consumption than high-performance liquid chromatography (HPLC) in the analysis of the same samples using the same type of stationary phase. Proper selection of the mixed mobile phases was critical for the separation of these complex mixtures with enhanced speed and selectivity. The use of a step gradient further improved the speed of the CEC analysis resulting in electrochromatograms that required only 25-70% of the corresponding HPLC analysis times.

DNA Adducts Formed by syn Dihydrodiol Epoxides of Polycyclic Aromatic Hydrocarbons

Abstract A brief review of the literature on syn dihydrodiol epoxide DNA adducts is presented. NMR studies indicate that bay region substituents influence the conformation of the partially saturated ring in such adducts. However, only substitutions that lead to distortion from planarity are associated with reactivity towards deoxyadenosine residues in DNA and with greater tumorigenicity. This is similar to the situation that obtains for anti dihydrodiol epoxides.

Quantitative reactions of anti 5,9-dimethylchrysene dihydrodiol epoxide with DNA and deoxyribonucleotides.

Native as well as denatured calf thymus DNA, deoxyguanylic and deoxyadenylic acid, respectively, were reacted with the racemic anti 5,9-dimethylchrysene dihydrodiol epoxide (5,9-DMCDE). The deoxyribonucleoside adducts were separated by HPLC and characterized by CD and NMR. Approximately 17% of the epoxide was trapped by native DNA and 76% of the adducts were derived from the RSSR enantiomer. The ratios of dAdo/dGuo modification in DNA were 14/86 and 19/81 for RSSR and SRRS enantiomers, respectively. By monitoring the product yields of anti 5,9-DMCDE with DNA and deoxyribonucleotides, we hoped to gain further insight into the factors responsible for deoxyguanosine adduct formation by 5-methylchrysene dihydrodiol epoxide (5-MCDE) compared to 5, 6-dimethylchrysene dihydrodiol epoxide (5,6-DMCDE). The adduct yields in deoxyribonucleotide reactions of 5,9-DMCDE were slightly higher than those from 5-MCDE. However, the reaction yields of 5, 9-DMCDE with DNA were lower than those with 5-MCDE in most cases, particularly for the cis and trans deoxyadenosine adducts. It seems that the 9-methyl group of 5,9-DMCDE significantly influences adduct formation with the deoxyadenosine residue in DNA in contrast to the 6-methyl group of 5,6-DMCDE. The 9-methyl group sterically decreases deoxyadenosine adduct yields more in reaction with native DNA than denatured DNA, but it has little effect on deoxyribonucleotide reactions. Adduct formation with deoxyguanosine residues in DNA by all three dihydrodiol epoxides correlate with their respective tumorigenic and mutagenic activities.

DNA Adducts from syn 7-Methylbenz[A]anthracene 3,4-Dihydrodiol 1,2-Epoxide

Abstract DNA adducts derived from reaction of the racemic bay region syn 7-methylbenz[a]anthracene 3,4-dihydrodiol 1,2-epoxide with calf thymus DNA in vitro were tentatively identified. Eight markers (four deoxyguanosine and four deoxyadenosine adducts) were obtained from separate reactions of the racemic syn dihydrodiol epoxide with deoxyguanylic and deoxyadenylic acids. The nucleoside of origin of individual DNA products was established by comparing HPLC retention times and UV spectra of DNA adducts with those of the adduct markers. All DNA adducts eluted with retention times and had UV spectra that corresponded to the purine nucleoside adducts. Circular dichroism spectra of the marker adducts allowed assignment of S and R configuration at C1, the site of attachment of the hydrocarbon residue to the nucleoside. The CD spectra were comprised of four pairs of spectra that were mirror images of one another. Each pair consisted of the two cis or two trans products resulting from epoxide ring opening of each...

DNA Adducts Formed by the Fjord-Region Dihydrodiol Epoxide of Benzo[S]Picene

Abstract Anti benzo[s]picene 9,10-dihydrodiol 11,12-epoxide was reacted with calf thymus DNA and with its constituent deoxyribonucleotides. The origin of DNA adducts was established by comparison of their HPLC retention times and UV spectra with those of the nucleotide-derived adduct-markers. The low binding of the dihydrodiol epoxide to DNA (2%) and to deoxyguanylic and deoxyadenylic acids precluded an NMR study, and assignment of cis or trans epoxide ring opening was made by analogy to other anti dihydrodiol epoxide adducts. The adduct ratio for dGuo/dAdo in DNA was 24/76 and the ratios of dGuo cis/dGuo trans and dAdo cis/dAdo trans were 40/60 and 6/94, respectively.

Effects of Polycyclic Aromatic Hydrocarbon Adducts with Deoxyguanosine and Deoxyadenosine in vivo and in vitro

Abstract Reactive metabolites from non-planar polycyclic aromatic hydrocarbon carcinogens react extensively with both deoxyadenosine and deoxyguanosine in DNA whereas those from planar molecules react predominantly only with deoxyguanosine. In vitro studies with single adducts in oligonucleotides showed that both types of adduct blocked primer extension and that the limited amount of nucleotide addition opposite the adduct varied with the polymerase, the sequence context of the adduct and the chemical structure of the adduct. When these same single adduct containing-oligonucleotides were introduced into a single-stranded vector that was allowed to replicate in Escherichia coli, the major events observed were blockage of replication, insertion of the correct nucleotide (i.e. T opposite an A adduct and C opposite a G adduct), and insertion of A opposite the adduct. In mouse skin, benzo[c]phenanthrene 4S,3R-dihydrodiol 2S,1R-epoxide initiated substantially more tumors per DNA adduct formed than did the other...