Formation Of Covalent DNA Adducts Research Articles

Nitroreduction: A Critical Metabolic Pathway for Drugs, Environmental Pollutants, and Explosives.

Nitro group containing xenobiotics include drugs, cancer chemotherapeutic agents, carcinogens (e.g., nitroarenes and aristolochic acid) and explosives. The nitro group undergoes a six-electron reduction to form sequentially the nitroso-, N-hydroxylamino- and amino-functional groups. These reactions are catalyzed by nitroreductases which, rather than being enzymes with this sole function, are enzymes hijacked for their propensity to donate electrons to the nitro group either one at a time via a radical mechanism or two at time via the equivalent of a hydride transfer. These enzymes include: NADPH-dependent flavoenzymes (NADPH: P450 oxidoreductase, NAD(P)H-quinone oxidoreductase), P450 enzymes, oxidases (aldehyde oxidase, xanthine oxidase) and aldo-keto reductases. The hydroxylamino group once formed can undergo conjugation reactions with acetate or sulfate catalyzed by N-acetyltransferases or sulfotransferases, respectively, leading to the formation of intermediates containing a good leaving group which in turn can generate a nitrenium or carbenium ion for covalent DNA adduct formation. The intermediates in the reduction sequence are also prone to oxidation and produce reactive oxygen species. As a consequence, many nitro-containing xenobiotics can be genotoxic either by forming stable covalent adducts or by oxidatively damaging DNA. This review will focus on the general chemistry of nitroreduction, the enzymes responsible, the reduction of xenobiotic substrates, the regulation of nitroreductases, the ability of nitrocompounds to form DNA adducts and act as mutagens as well as some future directions.

A Comprehensive Database for DNA Adductomics.

The exposure of human DNA to genotoxic compounds induces the formation of covalent DNA adducts, which may contribute to the initiation of carcinogenesis. Liquid chromatography (LC) coupled with high-resolution mass spectrometry (HRMS) is a powerful tool for DNA adductomics, a new research field aiming at screening known and unknown DNA adducts in biological samples. The lack of databases and bioinformatics tool in this field limits the applicability of DNA adductomics. Establishing a comprehensive database will make the identification process faster and more efficient and will provide new insight into the occurrence of DNA modification from a wide range of genotoxicants. In this paper, we present a four-step approach used to compile and curate a database for the annotation of DNA adducts in biological samples. The first step included a literature search, selecting only DNA adducts that were unequivocally identified by either comparison with reference standards or with nuclear magnetic resonance (NMR), and tentatively identified by tandem HRMS/MS. The second step consisted in harmonizing structures, molecular formulas, and names, for building a systematic database of 279 DNA adducts. The source, the study design and the technique used for DNA adduct identification were reported. The third step consisted in implementing the database with 303 new potential DNA adducts coming from different combinations of genotoxicants with nucleobases, and reporting monoisotopic masses, chemical formulas, .cdxml files, .mol files, SMILES, InChI, InChIKey and IUPAC nomenclature. In the fourth step, a preliminary spectral library was built by acquiring experimental MS/MS spectra of 15 reference standards, generating in silico MS/MS fragments for all the adducts, and reporting both experimental and predicted fragments into interactive web datatables. The database, including 582 entries, is publicly available (https://gitlab.com/nexs-metabolomics/projects/dna_adductomics_database). This database is a powerful tool for the annotation of DNA adducts measured in (HR)MS. The inclusion of metadata indicating the source of DNA adducts, the study design and technique used, allows for prioritization of the DNA adducts of interests and/or to enhance the annotation confidence. DNA adducts identification can be further improved by integrating the present database with the generation of authentic MS/MS spectra, and with user-friendly bioinformatics tools.

Human Tyrosyl-DNA Phosphodiesterase 1 Possesses Transphosphooligonucleotidation Activity With Primary Alcohols.

Human tyrosyl-DNA phosphodiesterase 1 (TDP1) belongs to the phospholipase D superfamily, whose members contain paired catalytic histidine and lysine residues within two conserved motifs and hydrolyze phosphodiester bonds. TDP1 is a DNA repair enzyme that processes 3′ DNA end blocking lesions and a wide range of synthetic DNA adducts as a substrate. TDP1 hydrolyzes DNA-adducts via two coordinated SN2 nucleophilic attacks mediated by the action of two histidine residues and leads to the formation of the covalent intermediate. Hydrolysis of this intermediate is proposed to be carried out by a water molecule that is activated by the His493 residue acting as a general base. It was known that phospholipase D enzymes are able to catalyze not only hydrolysis but also a transphosphatidylation reaction in the presence of primary alcohols in which they transfer the substrate to the alcohol instead of water. Here, we first demonstrated that TDP1 is able to undergo a “transphosphooligonucleotidation” reaction, transferring the substrate residue to the alcohol, thus inducing the formation of covalent DNA adducts with different primary alcohol residues. Such adducts can be accumulated in the conditions of high concentration of alcohol. We demonstrated that glycerol residue was efficiently cleaved from the 3′-end by TDP1 but not by its mutant form associated with the disease spinocerebellar ataxia with axonal neuropathy. Therefore, the second reaction step can be carried out not only by a water molecule but also by the other small nucleophilic molecules, e.g., glycerol and ethanol. Thus, in some cases, TDP1 can be regarded not only as a repair enzyme but also as a source of DNA damage especially in the case of mutation. Such damages can make a negative contribution to the stability of cell vitality.

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs <em>In Vitro</em> and Their Determination by <sup>32</sup>P-postlabeling

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs <em>In Vitro</em> and Their Determination by <sup>32</sup>P-postlabeling

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling.

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of carcinogenic processes. This covalent binding, which is considered the cause of tumorigenesis, is now evaluated as a central dogma of chemical carcinogenesis. Here, methods are described employing the reactions catalyzed by cytochrome P450 and additional biotransformation enzymes to investigate the potency of chemicals or drugs for their activation to metabolites forming these DNA adducts. Procedures are presented describing the isolation of cellular fractions possessing biotransformation enzymes (microsomal and cytosolic samples with cytochromes P450 or other biotransformation enzymes, i.e., peroxidases, NADPH:cytochrome P450 oxidoreductase, NAD(P)H:quinone oxidoreductase, or xanthine oxidase). Furthermore, methods are described that can be used for the metabolic activation of analyzed chemicals by these enzymes as well as those for isolation of DNA. Further, the appropriate methods capable of detecting and quantifying chemical/drug-derived DNA adducts, i.e., different modifications of the 32P-postlabeling technique and employment of radioactive-labeled analyzed chemicals, are shown in detail.

Kinetic analysis of and platinum(II) migration in the reactions of tetrazolato-bridged dinuclear platinum(II) complexes with nucleotides

The series of tetrazolato-bridged complexes with the formula [{cis‑Pt(NH3)2}2(μ-OH)(μ-5-H-tetrazolato-N1,N2)]2+ (5-H-X) or [{cis‑Pt(NH3)2}2(μ-OH)(μ-5-R-tetrazolato-N2,N3)]n+ (R=H (5-H-Y), CH3 (1), CH2COOCH2CH3 (2), CH2COO– (3), n=2 (5-H-Y, 1, 2) or 1 (3)) are promising candidate complexes for formulation as next-generation platinum-based anticancer drugs that form multimodal bindings with DNA molecules. These multimodal bindings involve both non-covalent and covalent interactions, the latter of which are acknowledged to be essential for platinum-based drugs to exert their anticancer activity. In the present study, the tetrazolato-bridged complexes reacted with two molar equivalents of guanosine-5′-monophosphate (GMP) to yield the 1:2 reaction products [{cis‑Pt(NH3)2(GMP-N7)}2(μ-5-R-tetrazolato-N1,N3)]2− or 1−. This reaction was accompanied by an intramolecular Pt(II) migration that contributed to the formation of diverse DNA crosslinking, such as interhelical crosslinks. The second-order reaction rate constants for the reactions performed in phosphate-buffered D2O solution showed that the reactivity of the complexes decreased in the order 5-H-X≳5-H-Y>2≳1>3 and that reactivity was correlated with the cytotoxicity of the complexes. A similar result was obtained for the reaction of the complexes with calf thymus DNA in which the formation of covalent DNA adducts was quantified by means of inductively coupled plasma mass spectrometry. These results suggest that overall charge affects the kinetics of the reactions of platinum complexes with GMP and calf thymus DNA. Thus, the positive charge of the complexes affects not only the non-covalent but also the covalent interactions between the complexes and nucleotides and DNA, which are negatively charged molecules.

Butyrate alters expression of cytochrome P450 1A1 and metabolism of benzo[a]pyrene via its histone deacetylase activity in colon epithelial cell models.

Butyrate, a short-chain fatty acid produced by fermentation of dietary fiber, is an important regulator of colonic epithelium homeostasis. In this study, we investigated the impact of this histone deacetylase (HDAC) inhibitor on expression/activity of cytochrome P450 family 1 (CYP1) and on metabolism of carcinogenic polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP), in colon epithelial cells. Sodium butyrate (NaBt) strongly potentiated the BaP-induced expression of CYP1A1 in human colon carcinoma HCT116 cells. It also co-stimulated the 7-ethoxyresorufin-O-deethylase (EROD) activity induced by the 2,3,7,8-tetrachlorodibenzo-p-dioxin, a prototypical ligand of the aryl hydrocarbon receptor. Up-regulation of CYP1A1 expression/activity corresponded with an enhanced metabolism of BaP and formation of covalent DNA adducts. NaBt significantly potentiated CYP1A1 induction and/or metabolic activation of BaP also in other human colon cell models, colon adenoma AA/C1 cells, colon carcinoma HT-29 cells, or in NCM460D cell line derived from normal colon mucosa. Our results suggest that the effects of NaBt were due to its impact on histone acetylation, because additional HDAC inhibitors (trichostatin A and suberanilohydroxamic acid) likewise increased both the induction of EROD activity and formation of covalent DNA adducts. NaBt-induced acetylation of histone H3 (at Lys14) and histone H4 (at Lys16), two histone modifications modulated during activation of CYP1A1 transcription, and it reduced binding of HDAC1 to the enhancer region of CYP1A1 gene. This in vitro study suggests that butyrate, through modulation of histone acetylation, may potentiate induction of CYP1A1 expression, which might in turn alter the metabolism of BaP within colon epithelial cells.

Abstract 826: DNA adducts induced by in vitro activation of diesel and biodiesel exhaust extracts

Abstract Petroleum diesel exhaust is a complex mixture containing many probable and known carcinogens. The development of biodiesel and biodiesel blends offers a renewable alternative to petroleum diesel, but few data are available concerning the carcinogenic potential of biodiesel exhausts. We compared the formation of covalent DNA adducts by the in vitro metabolic activation of organic extracts of diesel exhaust particles (DEP)from petroleum diesel and biodiesel. Two different petroleum diesel DEPs were examined (C-DEP and B0), as well as one biodiesel DEP (B100), and DEP resulting from combustion of a blend of 20% B100 and 80% B0 (B20), which is representative of commercially available biodiesel. Oxidative activation was performed in the presence of calf thymus DNA (ctDNA) using rat liver microsomal fractions with required cofactors (S9), and nitroreductive activation was performed using xanthine oxidase (XO) and hypoxanthine in the presence of ctDNA. The modified DNA was hydrolyzed and analyzed by 32P-postlabeling using either butanol extraction or nuclease P1 pre-enrichment. Multiple DNA adducts were produced with chromatographic mobilities consistent with polycyclic aromatic hydrocarbon (PAH) and nitro-PAH adducts. The types and quantities of DNA adducts produced from two independent petroleum diesel DEPs were similar, with evidence of formation of both PAH- and nitroPAH- derived adducts. In contrast, the biodiesel DEP B100 induced higher total levels of DNA adducts in both activation systems. The lowest levels of DNA adduct formation were observed with B20 DEP. These results suggest that the DEP from available biodiesel blends (B20) pose lower risk for induction of DNA damage than petroleum diesel. This is an abstract or a proposed presentation and does not necessarily reflect EPA policy. Citation Format: Jeffrey A. Ross, Garret B. Nelson, Esrta Mutlu, Sarah H. Warren, Matthews P. Peggy, M. Ian Gilmour, David M. DeMarini. DNA adducts induced by in vitro activation of diesel and biodiesel exhaust extracts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 826. doi:10.1158/1538-7445.AM2015-826

Inhibition of β-catenin signalling promotes DNA damage elicited by benzo[a]pyrene in a model of human colon cancer cells via CYP1 deregulation.

Deregulation of Wnt/β-catenin signalling plays an important role in the pathogenesis of colorectal cancer. Interestingly, this pathway has been recently implicated in transcriptional control of cytochrome P450 (CYP) family 1 enzymes, which are responsible for bioactivation of a number of dietary carcinogens. In the present study, we investigated the impact of inhibition of Wnt/β-catenin pathway on metabolism and genotoxicity of benzo[a]pyrene (BaP), a highly mutagenic polycyclic aromatic hydrocarbon and an efficient ligand of the aryl hydrocarbon receptor, which is known as a primary regulator of CYP1 expression, in cellular models derived from colorectal tumours. We observed that a synthetic inhibitor of β-catenin, JW74, significantly increased formation of BaP-induced DNA adducts in both colorectal adenoma and carcinoma-derived cell lines. Using the short interfering RNA (siRNA) targeting β-catenin, we then found that β-catenin knockdown in HCT116 colon carcinoma cells significantly enhanced formation of covalent DNA adducts by BaP and histone H2AX phosphorylation, as detected by (32)P-postlabelling technique and immunocytochemistry, respectively, and it also induced expression of DNA damage response genes, such as CDKN1A or DDB2. The increased formation of DNA adducts formed by BaP upon β-catenin knockdown corresponded with enhanced production of major BaP metabolites, as well as with an increased expression/activity of CYP1 enzymes. Finally, using siRNA-mediated knockdown of CYP1A1, we confirmed that this enzyme plays a major role in formation of BaP-induced DNA adducts in HCT116 cells. Taken together, the present results indicated that the siRNA-mediated inhibition of β-catenin signalling, which is aberrantly activated in a majority of colorectal cancers, modulated genotoxicity of dietary carcinogen BaP in colon cell model in vitro, via a mechanism involving up-regulation of CYP1 expression and activity.

Formation of DNA adducts by ellipticine and its micellar form in rats - a comparative study.

The requirements for early diagnostics as well as effective treatment of cancer diseases have increased the pressure on development of efficient methods for targeted drug delivery as well as imaging of the treatment success. One of the most recent approaches covering the drug delivery aspects is benefitting from the unique properties of nanomaterials. Ellipticine and its derivatives are efficient anticancer compounds that function through multiple mechanisms. Formation of covalent DNA adducts after ellipticine enzymatic activation is one of the most important mechanisms of its pharmacological action. In this study, we investigated whether ellipticine might be released from its micellar (encapsulated) form to generate covalent adducts analogous to those formed by free ellipticine. The 32P-postlabeling technique was used as a useful imaging method to detect and quantify covalent ellipticine-derived DNA adducts. We compared the efficiencies of free ellipticine and its micellar form (the poly(ethylene oxide)-block-poly(allyl glycidyl ether) (PAGE-PEO) block copolymer, P 119 nanoparticles) to form ellipticine-DNA adducts in rats in vivo. Here, we demonstrate for the first time that treatment of rats with ellipticine in micelles resulted in formation of ellipticine-derived DNA adducts in vivo and suggest that a gradual release of ellipticine from its micellar form might produce the enhanced permeation and retention effect of this ellipticine-micellar delivery system.

Enzymes Metabolizing Aristolochic Acid and their Contribution to the Development of Aristolochic Acid Nephropathy and Urothelial Cancer

Aristolochic acid (AA), a plant nephrotoxin and carcinogen, causes aristolochic acid nephropathy (AAN) and its associated urothelial malignancy, and is hypothesized to be responsible for Balkan endemic nephropathy (BEN). The major component of AA, aristolochic acid I (AAI), is the predominant compound responsible for these diseases. The reductive activation of AAI leads to the formation of covalent DNA adducts. The most abundant DNA adduct, 7-(deoxyadenosin-N6-yl)aristolactam I, causes characteristic AT→TA transversions found in the TP53 tumor suppressor gene in tumors from AAN and BEN patients. Understanding which human enzymes are involved in AAI activation to species forming DNA adducts and/or detoxication to the AAI O-demethylated metabolite, aristolochic acid Ia (AAIa), is important in the assessment of the susceptibility to this carcinogen. This review summarizes the latest data on identifying human and rodent enzymes participating in AAI metabolism. NAD(P)H:quinone oxidoreductase (NQO1) is the most efficient cytosolic nitroreductase activating AAI in vitro and in vivo. In human hepatic microsomes, AAI is activated by cytochrome P450 1A2 (CYP1A2) and, to a lesser extent, by CYP1A1; NADPH:CYP oxidoreductase also plays a minor role. Human and rodent CYP1A1 and 1A2 are also the principal enzymes involved in oxidative detoxication of AAI to AAIa in vitro and in vivo. The orientation of AAI in the active sites of human CYP1A1/2 and NQO1 was predicted from molecular modeling and is consistent with the efficient reduction of AAI by them observed experimentally. Molecular modeling also shows why CYP1A2 plays an important role in the oxidation of AAI to AAIa.

Genotoxicity of 2,6- and 3,5-dimethylaniline in cultured mammalian cells: the role of reactive oxygen species.

Several alkylanilines with structures more complex than toluidines have been associated epidemiologically with human cancer. Their mechanism of action remains largely undetermined, and there is no reported evidence that it replicates that of multicyclic aromatic amines even though the principal metabolic pathways of P450-mediated hydroxylation and phase II conjugation are very similar. As a means to elucidate their mechanisms of action, lethality and mutagenicity in the adenine phosphoribosyltransferase (aprt (+/-)) gene induced in several Chinese hamster ovary cell types by 2,6- and 3,5-dimethylaniline (2,6-DMA, 3,5-DMA) and their N- and ring-hydroxyl derivatives (N-OH-2,6-DMA, N-OH-3,5-DMA, 2,6-DMAP, 3,5-DMAP) were assessed. Dose-response relationships were determined in the parental AA8 cell line, its repair-deficient UV5 subclone and other repair-deficient 5P3NAT2 or -proficient 5P3NAT2R9 subclones engineered to express mouse cytochrome P4501A2 (CYP1A2) and human N-acetyltransferase (NAT2), and also in AS52 cells harboring the bacterial guanine-hypoxanthine phosphoribosyltransferase (gpt) gene. Mutations in the gpt gene of AS52 cells were characterized and found to be dominated by G:C to A:T and A:T to G:C transitions. Separately, treatment of AS52 cells with N-OH-2,6-DMA, N-OH-3,5-DMA, 2,6-DMAP, 3,5-DMAP, and 3,5-DMAP led to intracellular production of reactive oxygen species (ROS) for at least 24h after removal of the mutagens in every case. Using the comet assay, DNA strand breaks were observed in a dose-dependent manner in AS52 cells when treated with each of the four N-OH-2,6-DMA, N-OH-3,5-DMA, 2,6-DMAP, and 3,5-DMAP derivatives. Comparative evaluation of the results indicates that the principal mechanism of mutagenic action is likely to be through redox cycling of intracellularly bound aminophenol/quinone imine structures to generate ROS rather than through formation of covalent DNA adducts.

Analysis of covalent ellipticine- and doxorubicin-derived adducts in DNA of neuroblastoma cells by the <sup>32</sup>P-postlabeling technique

Ellipticine and doxorubicin are antineoplastic agents, whose action is based mainly on DNA damage such as intercalation, inhibition of topoisomerase II and formation of covalent DNA adducts. The key target to resolve which of these mechanisms are responsible for ellipticine and doxorubicin anticancer effects is the development of suitable methods for identifying their individual DNA-damaging effects. Here, the (32)P-postlabeling method was tested to detect covalent DNA adducts formed by ellipticine and doxorubicin. The standard procedure of (32)P-postlabeling assay, this procedure under ATP-deficient conditions, the version using extraction of adducts with n-butanol and the nuclease P1 enrichment version were used to analyze ellipticineand/ or doxorubicin-derived DNA adducts. Two covalent ellipticine-derived DNA adducts, which are associated with cytotoxicity of ellipticine to human UKF-NB-3 and UKF-NB-4 neuroblastoma cell lines, were detected by the (32)P-postlabeling method. These adducts are identical to those formed by the ellipticine metabolites, 13-hydroxy- and 12-hydroxyellipticine. In contrast, no covalent adducts formed by doxorubicin in DNA of these neuroblastoma cells and in DNA incubated with this drug and formaldehyde in vitro were detectable by the (32)P-postlabeling assay. The results presented in this paper are the first to demonstrate that in contrast to covalent DNA adducts formed by ellipticine, the adducts generated by formaldehyde-mediated covalent binding of doxorubicin to DNA are not detectable by the (32)P-postlabeling assay. No DNA adducts were, detectable either in vitro, in incubations of DNA with doxorubicin or in DNA of neuroblastoma cells treated with this drug. The results also suggest that covalent binding of ellipticine to DNA of UKF-NB-3 and UKF-NB-4 neuroblastoma cell lines is the predominant mechanism responsible for the cytotoxicity of this drug. To understand the mechanisms of doxorubicin anticancer effects on neuroblastoma cells, development of novel methods for identifying covalent doxorubicin-derived DNA adducts is the major challenge for further research.

An Update on Direct Genotoxicity as a Molecular Mechanism of Ochratoxin A Carcinogenicity

Ochratoxin A (OTA) is a naturally occurring chlorophenolic fungal toxin that contaminates a wide range of food products and poses a cancer threat to humans. The mechanism of action (MOA) for OTA renal carcinogenicity is a controversial issue. In 2005, direct genotoxicity (covalent DNA adduct formation) was proposed as a MOA for OTA-mediated carcinogenicity [ Manderville , R. A. ( 2005 ) Chem. Res. Toxicol. 18 , 1091 - 1097 ]. At that time, inconsistent results had been published on OTA genotoxicity/mutagenicity, and conclusive evidence for OTA-mediated DNA adduction had been lacking. In this update, published data from the past 6-7 years are presented that provide new hypotheses for the MOA of OTA-mediated carcinogenicity. While direct genotoxicity remains a controversial issue for OTA, new findings from the Umemura and Nohmi laboratories provide definitive results for the mutagenicity of OTA in the target tissue (outer medulla) of male rat kidney that rules out oxidative DNA damage. These findings, coupled with our own efforts that provide new structural evidence for DNA adduction by OTA, has strengthened the argument for involvement of direct genotoxicity in OTA-mediated renal carcinogenesis. This MOA should be taken into consideration for OTA human risk assessment.

Genotoxicity of Colloidal Fullerene C60

Previous genotoxicity tests of aqueous fullerene C₆₀) suspension (aqu-C₆₀) yielded both positive and negative results. In the present study, aqu-C₆₀ elicited positive responses in two bacterial genotoxicity tests, the Bacillus subtilis Rec-assay and the umu test at concentrations as low as 0.048 mg/L and 0.43 mg/L, respectively. In mammalian cell experiments, aqu-C₆₀ showed a significant growth inhibitory effect on human hepatocarcinoma HepG2 cells at 0.46 mg/L. The level of the oxidative DNA lesion 8-oxo-7,8-dihydro-2'-deoxyguanosine, measured by liquid chromatography tandem mass spectrometry, was slightly but not significantly increased in HepG2 cells treated with 0.46 mg/L for 24 h, whereas the level of the lipid peroxidation-related DNA lesion α-methyl-γ-hydroxy-1,N²)-propano-2'-deoxyguanosine was not changed. Under the same conditions, we did not detect any bulky DNA adducts, as measured by ³²P-postlabeling/polyacrylamide gel electrophoresis analysis. Our data suggest that aqu-C₆₀ has DNA-damaging potential and that the DNA damage is not due to covalent DNA adduct formation by C₆₀ itself.

The mechanism of cytotoxicity and DNA adduct formation by the anticancer drug ellipticine in human neuroblastoma cells

Ellipticine is an antineoplastic agent, whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of covalent DNA adducts mediated by cytochromes P450 and peroxidases. Here, the molecular mechanism of DNA-mediated ellipticine action in human neuroblastoma IMR-32, UKF-NB-3 and UKF-NB-4 cancer cell lines was investigated. Treatment of neuroblastoma cells with ellipticine resulted in apoptosis induction, which was verified by the appearance of DNA fragmentation, and in inhibition of cell growth. These effects were associated with formation of two covalent ellipticine-derived DNA adducts, identical to those formed by the cytochrome P450- and peroxidase-mediated ellipticine metabolites, 13-hydroxy- and 12-hydroxyellipticine. The expression of these enzymes at mRNA and protein levels and their ability to generate ellipticine-DNA adducts in neuroblastoma cells were proven, using the real-time polymerase chain reaction, Western blotting analyses and by analyzing ellipticine-DNA adducts in incubations of this drug with neuroblastoma S9 fractions, enzyme cofactors and DNA. The levels of DNA adducts correlated with toxicity of ellipticine to IMR-32 and UKF-NB-4 cells, but not with that to UKF-NB-3 cells. In addition, hypoxic cell culture conditions resulted in a decrease in ellipticine toxicity to IMR-32 and UKF-NB-4 cells and this correlated with lower levels of DNA adducts. Both these cell lines accumulated in S phase, suggesting that ellipticine-DNA adducts interfere with DNA replication. The results demonstrate that among the multiple modes of ellipticine antitumor action, formation of covalent DNA adducts by ellipticine is the predominant mechanism of cytotoxicity to IMR-32 and UKF-NB-4 neuroblastoma cells.

The comparison of cytotoxicity of the anticancer drugs doxorubicin and ellipticine to human neuroblastoma cells

Ellipticine is an antineoplastic agent, whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of covalent DNA adducts mediated by cytochromes P450 and peroxidases. Here, the cytotoxicity of ellipticine to human neuroblastoma derived cell lines IMR-32 and UKF-NB-4 was investigated. Treatment of neuroblastoma cells with ellipticine was compared with that of these cancer cells with doxorubicin. The toxicity of ellipticine was essentially the same as that of doxorubicin to UKF-NB-4 cells, but doxorubicin is much more effective to inhibit the growth of the IMR-32 cell line than ellipticine. Hypoxic conditions used for the cell cultivation resulted in a decrease in ellipticine and/or doxorubicin toxicity to IMR-32 and UKF-NB-4 neuroblastoma cells.

The Anticancer Drug Ellipticine Is a Potent Inducer of Rat Cytochromes P450 1A1 and 1A2, Thereby Modulating Its Own Metabolism

Ellipticine is an antineoplastic agent whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II, and formation of covalent DNA adducts mediated by cytochromes P450 (P450s) and peroxidases. Here, this drug was found to induce CYP1A1 and/or 1A2 enzymes and their enzymatic activities in livers, lungs, and kidneys of rats treated (i.p.) with ellipticine. The induction is transient. In the absence of repeated administration of ellipticine, the levels and activities of the induced CYP1A decreased almost to the basal level 2 weeks after treatment. The ellipticine-mediated CYP1A induction increases the DNA adduct formation by the compound. When microsomal fractions from livers, kidneys, and lungs of rats treated with ellipticine were incubated with ellipticine, DNA adduct formation, measured by (32)P-postlabeling analysis, was up to 3.8-fold higher in incubations with microsomes from pretreated rats than with controls. The observed stimulation of DNA adduct formation by ellipticine was attributed to induction of CYP1A1 and/or 1A2-mediated increase in ellipticine oxidative activation to 13-hydroxy- and 12-hydroxyellipticine, the metabolites generating two major DNA adducts in human and rat livers. In addition to these metabolites, increased formation of the excretion products 9-hydroxy- and 7-hydroxyellipticine was also observed in microsomes of rats treated with ellipticine. Taken together, these results demonstrate for the first time that by inducing CYP1A1/2, ellipticine increases its own metabolism, leading both to an activation of this drug to reactive species-forming DNA adducts and to detoxication metabolites, thereby modulating to some extent its pharmacological and/or genotoxic potential.

Mammalian peroxidases activate anticancer drug ellipticine to intermediates forming deoxyguanosine adducts in DNA identical to those found in vivo and generated from 12‐hydroxyellipticine and 13‐hydroxyellipticine

Ellipticine is a potent antineoplastic agent, whose mode of action is considered to be based mainly on DNA intercalation, inhibition of topoisomerase II and cytochrome P450-mediated formation of covalent DNA adducts. This is the first report on the molecular mechanism of ellipticine oxidation by peroxidases (human myeloperoxidase, human and ovine cyclooxygenases, bovine lactoperoxidase, horseradish peroxidase) to species forming ellipticine-DNA adducts. Using NMR spectroscopy, the structures of 2 ellipticine metabolites were identified; the major product is the ellipticine dimer, in which the 2 ellipticine skeletons are connected via N(6) of the pyrrole ring of one ellipticine molecule and C9 in the second one. The minor metabolite is ellipticine N(2)-oxide. Using (32)P-postlabeling and [(3)H]-labeled ellipticine, we showed that ellipticine binds covalently to DNA after its activation by peroxidases. The DNA adduct pattern induced by ellipticine consisted of a cluster of up to 4 adducts. The 2 adducts are indistinguishable from the 2 major adducts generated between deoxyguanosine in DNA and either 13-hydroxy- or 12-hydroxyellipticine or in rats treated with ellipticine, or if ellipticine was activated with human hepatic and renal microsomes. The results presented here are the first characterization of the peroxidase-mediated oxidative metabolites of ellipticine and we have proposed species, 2 carbenium ions, ellipticine-13-ylium and ellipticine-12-ylium, as reactive species generating 2 major DNA adducts seen in vivo in rats treated with ellipticine. The study forms the basis to further predict the susceptibility of human cancers to ellipticine.

Molecular mechanisms of antineoplastic action of an anticancer drug ellipticine.

Ellipticine is a potent antineoplastic agent exhibiting the multimodal mechanism of its action. This article reviews the mechanisms of predominant pharmacological and cytotoxic effects of ellipticine and shows the results of our laboratories indicating a novel mechanism of its action. The prevalent mechanisms of ellipticine antitumor, mutagenic and cytotoxic activities were suggested to be intercalation into DNA and inhibition of DNA topoisomerase II activity. We demonstrated a new mode of ellipticine action, formation of covalent DNA adducts mediated by its oxidation with cytochromes P450 (CYP) and peroxidases. The article reports the molecular mechanism of ellipticine oxidation by CYPs and identifies human and rat CYPs responsible for ellipticine metabolic activation and detoxication. It also presents a role of peroxidases (i.e. myeloperoxidase, cyclooxygenases, lactoperoxidase) in ellipticine oxidation leading to ellipticine-DNA adducts. The 9-hydroxy- and 7-hydroxyellipticine metabolites formed by CYPs and the major product of ellipticine oxidation by peroxidases, the dimer, in which the two ellipticine skeletons are connected via N(6) of the pyrrole ring of one ellipticine molecule and C9 in the second one, are the detoxication metabolites. On the contrary, 13-hydroxy- and 12-hydroxyellipticine, produced by ellipticine oxidation with CYPs, the latter one formed also spontaneously from another CYP- and peroxidase-mediated metabolite, ellipticine N(2)-oxide, are metabolites responsible for formation of two ellipticine-derived deoxyguanosine adducts in DNA. The results reviewed here allow us to propose species, two carbenium ions, ellipticine-13-ylium and ellipticine-12-ylium, as reactive species generating two major DNA adducts seen in vivo in rats treated with ellipticine. The study forms the basis to further predict the susceptibility of human cancers to ellipticine.