Identification and quantitation of the depurination and stable DNA adducts of 7,12-dimethylbenz[a]anthracene (DMBA) formed by cytochrome P450 in rat liver microsomes previously established one-electron oxidation as the predominant mechanism of activation of DMBA to bind to DNA. In this paper we report the identification and quantitation of the depurination and stable DMBA-DNA adducts formed in mouse skin. The depurination adducts, which constitute 99% of all the adducts detected, are DMBA bound at the 12-methyl group to the N-7 of adenine or guanine, namely, 7-methylbenz[a]anthracene (MBA)-12-CH2-N7Ade and 7-MBA-12-CH2-N7Gua. The depurination adducts were identified by HPLC and fluorescence line narrowing spectroscopy. The stable DNA adducts were analyzed by the 32P-postlabeling method. Almost 4 times as much of the depurination adduct 7-MBA-12-CH2-N7Ade (79%) was formed compared to 7-MBA-12-CH2-N7Gua (20%). The stable adducts accounted for only 1% of all the adducts detected and 80% of these were formed from DMBA diolepoxide. The binding of DMBA to DNA specifically at the 12-CH3 group is consistent with the results of carcinogenicity experiments in which this group plays a key role. When DMBA was bound to RNA or denatured DNA in reactions catalyzed by microsomes or by horseradish peroxidase (HRP), no depurination DNA adducts of DMBA were detected. The amount of stable DNA adducts observed with denatured DNA was 70% lower in the HRP system and 30% lower in the microsomal system compared to native DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

S-(1-Acetoxymethyl)glutathione (GSCH(2)OAc) was synthesized and used as a model for the reaction of glutathione (GSH)-dihaloalkane conjugates with nucleosides and DNA. Previously, S-[1-(N(2)-deoxyguanosinyl)methyl]GSH had been identified as the major adduct formed in the reaction of GSCH(2)OAc with deoxyguanosine. GSCH(2)OAc was incubated with the three remaining deoxyribonucleosides to identify other possible adducts. Adducts to all three nucleosides were found using electrospray ionization mass spectrometry (ESI MS). The adduct of GSCH(2)OAc and deoxyadenosine was formed in yield of up to 0.05% and was identified as S-[1-(N(7)-deoxyadenosinyl)methyl]GSH. The pyrimidine deoxyribonucleoside adducts were formed more efficiently, resulting in yields of 1 and 2% for the GSCH(2)OAc adducts derived from thymidine and deoxycytidine, respectively, but their lability prevented their structural identification by (1)H NMR. On the basis of the available UV spectra, we propose the structures S-[1-(N(3)-thymidinyl)methyl]GSH and S-[1-(N(4)-deoxycytidinyl)methyl]GSH. Because adduct degradation occurred most rapidly at alkaline and neutral pH values, an enzymatic DNA digestion procedure was developed for the rapid hydrolysis of DNA to deoxyribonucleosides at acidic pH. DNA digests were completed in less than 2 h with a two-step method, which consisted of a 15 min incubation of DNA with high concentrations of deoxyribonuclease II and phosphodiesterase II at pH 4.5, followed by incubation of resulting nucleotides with acid phosphatase. Analysis of the hydrolysis products by HPLC-ESI-MS indicated the presence of the thymidine adduct.

Rabbit liver cytochrome P450 (P450) 1A2 was found to catalyze the 5,6-epoxidation of ␣-naphthoflavone (␣NF), 1-hydroxylation of pyrene, and the subsequent 6-, 8-, and other hydroxylations of 1-hydroxy (OH) pyrene. Plots of steady-state rates of product formation versus substrate concentration were hyperbolic for ␣NF epoxidation but highly cooperative (Hill n coefficients of 2-4) for pyrene and 1-OH pyrene hydroxylation. When any of the three substrates (␣NF, pyrene, 1-OH pyrene) were mixed with ferric P450 1A2 using stopped-flow methods, the changes in the heme Soret spectra were relatively slow and multiphasic. Changes in the fluorescence of all of the substrates were much faster, consistent with rapid initial binding to P450 1A2 in a manner that does not change the heme spectrum. For binding of pyrene to ferrous P450 1A2, the course of the spectra revealed sequential changes in opposite directions, consistent with P450 1A2 being involved in a series of transitions to explain the kinetic multiphasicity as opposed to multiple, slowly interconverting populations of enzyme undergoing the same event at different rates. Models of rabbit P450 1A2 based on a published crystal structure of a human P450 1A2-␣NF complex show active site space for only one ␣NF or for two pyrenes. The spectral changes observed for binding and hydroxylation of pyrene and 1-OH pyrene could be fit to a kinetic model in which hydroxylation occurs only when two substrates are bound. Elements of this mechanism may be relevant to other cases of P450 cooperativity.

The influences of base sequence on the thermodynamic properties associated with the interaction of actinomycin D with DNA are examined. It has been previously established that GpC steps of double-helical DNAs are highly preferred binding sites for actinomycin D. In this study, a series of oligonucleotides was designed and synthesized to probe the effects of flanking base sequence (adjacent to the GpC step) and novel non-GpC binding sites on the binding of actinomycin D. The use of these oligonucleotides provides a direct method for quantitating sequence specificities and actinomycin D binding energetics. Effects of different 5' and 3' flanking nucleotides on the interactions of actinomycin with the core GpC binding sites were examined using UV-visible spectrophotometric methods, and changes in binding energetics were quantitated. These studies demonstrate strong actinomycin D binding affinities to both classical GpC and an atypical non-GpC site. Enthalpy and entropy components of the DNA binding energetics for the GpC binding sites are compared and correlated with those determined for actinomycin D binding to the high-affinity non-GpC site of an 11-mer containing TGGGT as the central sequence. This TGGGT site, first suggested to be a high-affinity sequence in our earlier photoaffinity labeling studies, exhibits binding of actinomycin D comparable in strength to that of traditional actinomycin D binding sites (i.e., GpC steps). From these studies, the overall affinity and specific thermodynamic contributions (delta H degree, delta S degree) to binding of actinomycin D are demonstrated to be highly influenced both by the sequence at the intercalation site and by neighboring bases which flank the intercalation site.

Actinomycin D (ActD) is a DNA-binding antitumor antibiotic that appears to act in vivo by inhibiting RNA polymerase. The mechanism of DNA binding of ActD has attracted much attention because of its strong preference for 5'-dGpdC-3' sequences. Binding is thought to involve intercalation of the tricyclic aromatic phenoxazone ring into a GC step, with the two equivalent cyclic pentapeptide lactone substituents lying in the minor groove and making hydrogen bond contacts with the 2-amino groups of the nearest neighbor guanines. Recent studies have indicated, however, that binding is also influenced by next-nearest neighboring bases. We have examined this higher order specificity using 7-azido-actinomycin-D as a photoaffinity probe, and DNA sequencing techniques to quantitatively monitor sites of covalent photoaddition. We found that GC doublets were strongly preferred only if the 5'-flanking base was a pyrimidine and the 3'-flanking base was not cytosine. In addition we observed a previously unreported preference for binding at a GG doublet in the sequence 5'-TGGG-3'.