Formation Of Stable DNA Adducts Research Articles

Inflammatory mediators accelerate metabolism of benzo[a]pyrene in rat alveolar type II cells: The role of enhanced cytochrome P450 1B1 expression

Long-term deregulated inflammation represents one of the key factors contributing to lung cancer etiology. Previously, we have observed that tumor necrosis factor-α (TNF-α), a major pro-inflammatory cytokine, enhances genotoxicity of benzo[a]pyrene (B[a]P), a highly carcinogenic polycyclic aromatic hydrocarbon, in rat lung epithelial RLE-6TN cells, a model of alveolar type II cells. Therefore, we analyzed B[a]P metabolism in RLE-6TN cells under inflammatory conditions, simulated using either recombinant TNF-α, or a mixture of inflammatory mediators derived from activated alveolar macrophage cell line. Inflammatory conditions significantly accelerated BaP metabolism, as evidenced by decreased levels of both parent B[a]P and its metabolites. TNF-α altered production of the metabolites associated with dihydrodiol-epoxide and radical cation pathways of B[a]P metabolism, especially B[a]P-dihydrodiols, and B[a]P-diones. We then evaluated the role of cytochrome P450 1B1 (CYP1B1), which is strongly up-regulated in cells treated with B[a]P under inflammatory conditions, in the observed effects. The siRNA-mediated CYP1B1 knock-down increased levels of B[a]P and reduced formation of stable DNA adducts, thus confirming the essential role of CYP1B1 in B[a]P metabolism under inflammatory conditions. TNF-α also reduced expression of aldo-keto reductase 1C14, which may compete with CYP1B1 for B[a]P-7,8-dihydrodiol and divert it from the formation of ultimate B[a]P dihydrodiol epoxide. Together, the present data suggests that the CYP1B1-catalyzed metabolism of polycyclic aromatic hydrocarbons might contribute to their enhanced bioactivation and genotoxic effects under inflammatory conditions.

Platinum-based drugs and proteins: Reactivity and relevance to DNA adduct formation

The mechanism of action of clinically used Pt-based drugs is through the formation of stable DNA adducts occurring at the nitrogen in position 7 of guanine (N7) and involving one or two spatially closed residues. Nevertheless, proteins can represent alternative targets since in particular sulfur groups, present in cysteine or methionine residues, can efficiently coordinate platinum. Here we have characterized the reactivity profile of cisplatin, transplatin and of two trans-platinum amine derivatives (TPAs) towards three different proteins, bovine α-lactalbumin (α-LA), hen egg lysozyme (LYS) and human serum albumin (HSA). Our results demonstrate that generally the tested metal complexes react with the selected target causing protein oligomerization, likely through a cross-linking reaction. Interestingly, the extent of such a process is largely modulated by the target protein and by the chemical features of the metal complex, TPAs being the most efficient platinating agents. From a structural point of view the resulting reaction products turned out to be depending on the nature of the metal complexes. However, in all instances, a transfer reaction of the metal complex to DNA can also occur, maintaining the relevance of nucleic acids as a biological target. These results can be used to better rationalize the different pharmacological profiles reported for cisplatin and TPAs and can help in designing more predictive SARs within the series.

Solution Structure of a Covalently Bound Pyrrolo[2,1-c][1,4]benzodiazepine−Benzimidazole Hybrid to a 10mer DNA Duplex

A pyrrolo[2,1-c][1,4]benzodiazepine-benzimidazole hybrid (PBD-BIMZ) derived from the tricyclic anticancer PBD antibiotics can covalently bind to a guanine base at its exocyclic 2-amino group in double-helical DNA. Through the formation of stable DNA adducts, these hybrids have previously been shown to have significant anticancer activity in a number of cell lines. Here, the three-dimensional solution structure of the complex formed between the self-complementary DNA decamer 5'-AACAATTGTT-3' and PBD-BIMZ has been investigated by two-dimensional NMR spectroscopy and NOE distance restraint molecular dynamics simulations. Refinements using an explicit solvation model yielded a complex structure that is in good agreement with the NMR structural data. Successful convergence is indicated by an average mutual root-mean-square deviation of <1 A for three final representative structures selected by clustering methods from the molecular dynamics trajectories at 300 K. The ligand binds in an (11S,11aS) configuration to one of the two symmetrically located guanine bases of the duplex and is oriented with its benzimidazole moiety toward the 5'-end of the modified guanine. It is accommodated within the minor groove covering the centrally located 6 bp. Conformational and helical parameters of the DNA adduct are typical of a B-like duplex, and more significant helical distortions by the covalent binding of PBD-BIMZ are mostly confined to the covalent binding site and the junction between complexed and noncomplexed DNA segments. In contrast to the overall well-determined conformation of the bound hybrid, its terminal N-methylpiperazine ring appears to adopt various conformations associated with increased flexibility.

Formation of Stable DNA Adducts and Apurinic Sites upon Metabolic Activation of Bay and Fjord Region Polycyclic Aromatic Hydrocarbons in Human Cell Cultures

Carcinogenic polycyclic aromatic hydrocarbons (PAH), such as benzo[a]pyrene (B[a]P), 7,12-dimethylbenz[a]anthracene (DMBA), and dibenzo[a,l]pyrene (DB[a,l]P), are metabolically activated to electrophilically reactive bay or fjord region diol epoxides that bind to the exocyclic amino groups of purine bases in DNA to form stable adducts. In addition, it has been reported that these PAH can be enzymatically oxidized to yield radical cations that form apurinic (AP) sites in DNA via depurinating adducts. The formation of stable adducts and AP sites in DNA of human cells exposed to PAH was examined in cytochrome P450 (P450)-expressing mammary carcinoma MCF-7 cells and in leukemia HL-60 cells, which display a high peroxidase but no P450-mediated activity, after exposure to these PAH. Stable DNA adducts were assessed by (33)P-postlabeling/HPLC analysis, and the induction of AP sites in DNA was analyzed by an aldehyde reactive probe (ARP) and a slot blot method. After exposure for 4 h, the levels of stable DNA adducts were comparable in MCF-7 cells treated with B[a]P and DMBA, but significantly lower than those observed in MCF-7 cells treated with the stronger carcinogen DB[a,l]P. While the levels of stable adducts increased more than 10-fold (B[a]P and DMBA) or 100-fold (DB[a,l]P) after exposure for 24 h, the levels of AP sites remained low after both treatment periods. Thus, the levels of stable adducts were approximately 5-fold higher than the levels of AP sites after treatment with B[a]P or DMBA and more than 100-fold higher in cells exposed to DB[a,l]P for 24 h. None of these carcinogenic PAH formed detectable levels of stable DNA adducts or AP sites in HL-60 cells. The results demonstrate that metabolic activation of B[a]P, DMBA, and DB[a,l]P is catalyzed by P450 enzymes leading to diol epoxides that form predominantly stable DNA adducts but only low levels of AP sites.

Cancer initiation by polycyclic aromatic hydrocarbons results from formation of stable DNA adducts rather than apurinic sites.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants with high carcinogenic potencies that have been linked to the etiology of human cancers through their presence in cigarette smoke and environmental mixtures. They are metabolically activated in cells by cytochrome P450 enzymes and/or peroxidases to reactive intermediates that damage DNA. One pathway of activation forms dihydrodiol epoxides that covalently bind to exocyclic amino groups of purines in DNA to form stable adducts. Another pathway involves formation of radical cations that bind to the N7 or C8 of purines to form unstable adducts that depurinate to leave apurinic (AP) sites in DNA. In the present study the proportions of stable DNA adducts and AP sites formed by the carcinogenic PAHs dibenzo[a,l]-pyrene (DB[a,l]P), 7,12-dimethylbenz[a]anthracene (DMBA), and benzo[a]pyrene (B[a]P) have been investigated in a target tissue for carcinogenesis, mouse epidermis. After topical application of the PAHs on the skin of female SENCAR mice epidermal DNA was isolated and the formation of stable DNA adducts was measured by (33)P-postlabeling and HPLC analysis. AP sites in DNA were measured with an aldehyde reactive probe in a slot-blot assay. At both 4 and 24 h after exposure, DB[a,l]P formed significantly higher amounts of stable DNA adducts than DMBA, and B[a]P exhibited the lowest level of binding. In contrast, the number of AP sites present in mice treated with these PAHs was in the order: DMBA > B[a]P >> DB[a,l]P. The level of AP sites was significantly lower than the level of stable adducts for each PAH. The most potent carcinogen, DB[a,l]P, induced the highest level of stable adducts and the lowest level of AP sites in epidermal DNA. These results indicate that stable DNA adducts rather than AP sites are responsible for tumor initiation by carcinogenic PAHs.