Enzymatic Oxidation Of Drugs To Toxic And Carcinogenic M

Diabetes, Digestive, Kidney Diseases

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Abstract

Diol epoxide (DE) metabolites on benzo-rings of carcinogenic polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) and benzo[c]phenanthrene (BcPh) are believed to initiate cell transformation by covalent modification of DNA. Two diastereomeric DEs, each of which exists as a pair of enantiomers, are formed metabolically in mammals: DE-1, in which the benzylic hydroxyl group and epoxide oxygen are cis, and DE-2, in which these substituents are trans. The primary targets in DNA for these DEs are the exocyclic N-2 and N-6 amino groups of deoxyguanosine (dG) and deoxyadenosine (dA), respectively. We are using specific DE-DNA adducts as tools for probing the structural biology and mechanistic enzymology of enzymes required for essential cellular functions involving DNA processing. The goal is to elucidate the relationships between the physical structures of these DNA adducts and their biochemical processing in intact cells and with purified enzymes. Studies in the past year have focused on (1) the mutational consequences of replication of these adducts in M13mp7L2 constructs by SOS-induced Escherichia coli, and (2) the ways in which individual exonucleases, which hydrolyze the phosphodiester bonds in single-stranded oligonucleotides from either their 5'- or the 3'-ends, respond upon encountering hydrocarbon adducts. (1) In previous Reports we described the mutations induced in an E. Coli-M13 system by each of the eight possible adducts derived from cis and trans ring opening of the four optically active BaP 7,8-diol 9,10-epoxides by the N-6 amino group of a specific deoxyadenosine (dA) residue. BaP DE adducts formed by opening of the DEs by dA have three hydroxyl groups at C-7, -8, and -9 on the tetrahydrobenzo-ring and the purine N-6 at the fourth (C-10) position. Observed mutational frequencies depended in part on the relative spatial orientations of the three hydroxyl groups. To determine how the presence or absence of these hydroxyl groups affects mutational response, 16-mer oligonucleotides were synthesized with a sequence previously studied with the BaP DE-dA adducts, but containing dA adducts in which two or all three of the hydroxyl groups were replaced by a hydrogen. These 16-mers were incorporated into the M13 DNA and allowed to replicate in SOS-induced E. coli. In general, decreasing the number of adduct hydroxyl groups decreased the total frequency of substitution mutations. For all but one of the present adducts, the total mutational frequency was lower than that for any of the previously reported DE adducts in the same sequence. The exception was the 10R diastereomer of the adduct derived from cis opening of 9,10-epoxy-7,8,9,10-tetrahydro BaP at C-10 by the N-6 amino group of dA, which gave the highest mutational frequency of all the BaP dA adducts studied to date in this DNA sequence, including the DE adducts. In general, adducts derived from cis opening of the epoxide ring of the above tetrahydroepoxide as well as of the diol epoxides were more mutagenic than the corresponding trans adducts. With the present set of BaP dA adducts with one or no hydroxyl groups on the tetrahydrobenzo-ring, A to T transversions predominated, with smaller numbers of A to G transitions and even fewer A to C transversions. (2) Snake venom phosphodiesterase (VPD) and bovine spleen phosphodiesterase (SPD) are exonucleases that cleave single-stranded oligonucleotides in opposite directions (from the 3'- to 5'- and the 5'- to 3'-ends, respectively). We investigated the ease of hydrolysis by VPD and SPD of short deoxyoligonucleotides containing defined BaP or BcPh DE adducts at N-6 of dA. In accordance with earlier reports, adducts with R configuration at the site of attachment of dA to the hydrocarbon moiety are generally more resistant to hydrolysis by VPD than their S-diastereomers. Furthermore, adducts derived from cis opening of the epoxide ring are considerably more resistant to VPD than the corresponding trans-opened adducts. Although several previous investigations had suggested that oligonucleotides containing adducts with S configuration at the site of attachment of dA to the hydrocarbon are more resistant to cleavage by SPD than are their R-diastereomers, our present results with a more extensive set of oligonucleotides indicate that SPD, in contrast to VPD, exhibits little discrimination between adducts with R and S configuration. In the case of VPD, the exonucleolytic hydrolysis of oligonucleotides containing either R- or S-adduct diastereomers initially yields a fragment containing the adducted dA residue at its 3'-end. The enzyme then "skips" over the next, resistant bond to give a dimer (pNpA*) with an intact 5'-phosphodiester bond to the adducted dA*. With several of the adducts, this dimer eventually undergoes slower cleavage to release the monomer, p(dA*). In the case of SPD, the enzyme stalls to give a fragment containing one unmodified base 5' to the adduct (NpA*pNpNpN.....). Although this intermediate can eventually be slowly hydrolyzed to the nucleotide level, "skipping" of the resistant bond to give a dimer is not observed with SPD. A striking observation is that for both enzymes, the most resistant internucleotide linkage is between the modified base and the base immediately 5' to it, regardless of the configuration of the adduct. This suggests that both R- and S-dA adducts are "seen" by VPD and SPD as qualitatively blocking access to the same side of the modified base. These results with BaP and BcPh DE-dA adducts contrast with observations by other investigators with BaP DE-dG adducts, in which the resistant internucleotide linkage is dependent on adduct configuration, and is immediately 5' to S-dG adducts and immediately 3' to R-dG adducts.