Induction of AT-specific DNA-interstrand crosslinks by bizelesin in genomic and simian virus 40 DNA.
Bizelesin is a bifunctional AT-specific DNA alkylating drug. Our study characterized the ability of bizelesin to induce interstrand crosslinks, a potential lethal lesion. In genomic DNA of BSC-1 cells, bizelesin formed from approx. 0.3 to 6.03+/-0.85 interstrand crosslinks per 106 base pairs, at 5-100 nM drug concentration, respectively, comparable to the number of total adducts previously determined in the same system (J.M. Woynarowski, M.M. McHugh, L.S. Gawron, T.A. Beerman, Biochemistry 34 (1995) 13042-13050). Bizelesin did not induce DNA-protein crosslinks or strand breaks. A model defined target, intracellular simian virus 40 (SV40) DNA, was employed to map at the nucleotide level sites of bizelesin adducts, including potential interstrand crosslinks. Preferential adduct formation was observed at AT tracts which are abundant in the SV40 matrix associated region and the origin of replication. Many sites, including each occurrence of 5'-T(A/T)4A-3', co-mapped on both DNA strands suggesting interstrand crosslinks, although monoadducts were also formed. Bizelesin adducts in naked SV40 DNA were found at similar sites. The localization of bizelesin-induced crosslinks in AT-rich tracts of replication-related regions is consistent with the potent anti-replicative properties of bizelesin. Given the apparent lack of other types of lesions in genomic DNA, interstrand crosslinks localized in AT-rich tracts, and to some extent perhaps also monoadducts, are likely to be lethal effects of bizelesin. (+info)
The effect of cotinine or cigarette smoke co-administration on the formation of O6-methylguanine adducts in the lung and liver of A/J mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, induces lung adenomas in A/J mice, following a single intraperitoneal (i.p.) injection. However, inhalation of tobacco smoke has not induced or promoted tumors in these mice. NNK-induced lung tumorigenesis is thought to involve O6-methylguanine (O6MeG) formation, leading to GC-->AT transitional mispairing and an activation of the K-ras proto-oncogene in the A/J mouse. NNK can be metabolized by several different cytochromes P450, resulting in a number of metabolites. Formation of the promutagenic DNA adduct O6MeG is believed to require metabolic activation of NNK by cytochrome P450-mediated alpha-hydroxylation of the methylene group adjacent to the N-nitroso nitrogen to yield the unstable intermediate, methanediazohydroxide. Nicotine, cotinine (the major metabolite of nicotine), and aqueous cigarette tar extract (ACTE) have all been shown to effectively inhibit metabolic activation of NNK to its mutagenic form, most likely due to competitive inhibition of the cytochrome P450 enzymes involved in alpha-hydroxylation of NNK. The objective of the current study was to monitor the effects of cotinine and cigarette smoke (CS) on the formation of O6MeG in target tissues of mice during the acute phase of NNK treatment. To test the effect of cotinine, mature female A/J mice received a single intraperitoneal injection of NNK (0, 2.5, 5, 7.5, or 10 mumole/mouse) with cotinine administered at a total dose of 50 mumole/mouse in 3 separate i.p. injections, administered 30 min before, immediately after, and 30 min after NNK treatment. To test the effect of whole smoke exposure on NNK-related O6MeG formation, mice were exposed to smoke generated from Kentucky 1R4F reference cigarettes at 0, 0.4, 0.6, or 0.8 mg wet total particulate matter/liter (WTPM/L) for 2 h, with a single i.p. injection of NNK (0, 3.75, or 7.5 mumole/mouse) midway through the exposure. Cigarette smoke alone failed to yield detectable levels of O6MeG. The number of O6MeG adducts following i.p. injection of NNK was significantly (p < 0.05) reduced in both lung and liver by cotinine and by cigarette smoke exposure. Our results demonstrate that NNK-induced O6MeG DNA adducts in A/J mice are significantly reduced when NNK is administered together with either cotinine, the major metabolite of nicotine, or the parental complex mixture, cigarette smoke. (+info)
Effect of cellular ATP depletion on topoisomerase II poisons. Abrogation Of cleavable-complex formation by etoposide but not by amsacrine.
Topoisomerase (topo) II poisons have been categorized into ATP-independent and -dependent drugs based on in vitro studies. We investigated drug-induced topoII-DNA complexes in intact cells almost completely depleted of ATP. Virtually no DNA single-strand breaks (SSBs), as measured by alkaline elution, were detected in energy-depleted cells treated with the topoII poisons etoposide, teniposide, daunorubicin, doxorubicin, mitoxantrone, or clerocidin. This inhibition was reversible; subsequent incubation with glucose restored the level of DNA SSBs. The effect of ATP depletion was specific for topoII, because topoI-mediated cleavable complexes induced by camptothecin were unaffected by ATP depletion. Furthermore, etoposide-induced DNA-protein complexes and DNA double-strand breaks, as measured by filter elution techniques, and topoIIalpha and -beta trapping, as measured by a band depletion assay, were completely inhibited by energy depletion. Differences in drug transport could not explain the effect of ATP depletion. The topoII poison amsacrine (m-AMSA) was unique with respect to ATP dependence. In ATP-depleted cells, m-AMSA-induced DNA SSBs, DNA double-strand breaks, DNA-protein complexes, topoIIalpha and -beta trapping were only modestly reduced. The accumulation of m-AMSA was reduced in ATP-depleted cells, which indicates that drug transport could contribute to the modest decrease in m-AMSA-induced cleavable complexes. In conclusion, drug-induced topoII-DNA complexes were completely antagonized in ATP-depleted cells, except in the case of m-AMSA. One possible interpretation is that m-AMSA mainly produces prestrand passage DNA lesions, whereas the other topoII poisons tested exclusively stabilize poststrand passage DNA lesions in intact cells. (+info)
A novel trinuclear platinum complex overcomes cisplatin resistance in an osteosarcoma cell system.
Multinuclear platinum compounds have been designed to circumvent the cellular resistance to conventional platinum-based drugs. In an attempt to examine the cellular basis of the preclinical antitumor efficacy of a novel multinuclear platinum compound (BBR 3464) in the treatment of cisplatin-resistant tumors, we have performed a comparative study of cisplatin and BBR 3464 in a human osteosarcoma cell line (U2-OS) and in an in vitro selected cisplatin-resistant subline (U2-OS/Pt). A marked increase of cytotoxic potency of BBR 3464 in comparison with cisplatin in U2-OS cells and a complete lack of cross-resistance in U2-OS/Pt cells were found. A detailed analysis of the cisplatin-resistant phenotype indicated that it was associated with reduced cisplatin accumulation, reduced interstrand cross-link (ICL) formation and DNA platination, microsatellite instability, and reduced expression of the DNA mismatch repair protein PMS2. Despite BBR 3464 charge and molecular size, in U2-OS and U2-OS/Pt cells, BBR 3464 accumulation and DNA-bound platinum were much higher than those observed for cisplatin. In contrast, the frequency of ICLs after exposure to BBR 3464 was very low. The time course of ICL formation after drug removal revealed a low persistence of these types of DNA lesions induced by BBR 3464, in contrast to an increase of DNA lesions induced by cisplatin, suggesting that components of the DNA repair pathway handle the two types of DNA lesions differently. The cellular response of HCT116 mismatch repair-deficient cells was consistent with a lack of influence of mismatch repair status on BBR 3464 cytotoxicity. Because BBR 3464 produces high levels of lesions different from ICLs, likely including intra-strand cross-links and monoadducts, the ability of the triplatinum complex to overcome cisplatin resistance appears to be related to a different mechanism of DNA interaction (formation of different types of drug-induced DNA lesions) as compared with conventional mononuclear complexes rather than the ability to overcome specific cellular alterations. (+info)
Biomarkers for exposure to ambient air pollution--comparison of carcinogen-DNA adduct levels with other exposure markers and markers for oxidative stress.
Human exposure to genotoxic compounds present in ambient air has been studied using selected biomarkers in nonsmoking Danish bus drivers and postal workers. A large interindividual variation in biomarker levels was observed. Significantly higher levels of bulky carcinogen-DNA adducts (75.42 adducts/10(8) nucleotides) and of 2-amino-apidic semialdehyde (AAS) in plasma proteins (56.7 pmol/mg protein) were observed in bus drivers working in the central part of Copenhagen, Denmark. In contrast, significantly higher levels of AAS in hemoglobin (55.8 pmol/mg protein), malondialdehyde in plasma (0. 96 nmol/ml plasma), and polycyclic aromatic hydrocarbon (PAH)-albumin adduct (3.38 fmol/ microg albumin) were observed in the suburban group. The biomarker levels in postal workers were similar to the levels in suburban bus drivers. In the combined group of bus drivers and postal workers, negative correlations were observed between bulky carcinogen-DNA adduct and PAH-albumin levels (p = 0.005), and between DNA adduct and [gamma]-glutamyl semialdehyde (GGS) in hemoglobin (p = 0.11). Highly significant correlations were found between PAH-albumin adducts and AAS in plasma (p = 0.001) and GGS in hemoglobin (p = 0.001). Significant correlations were also observed between urinary 8-oxo-7, 8-dihydro-2'-deoxyguanosine and AAS in plasma (p = 0.001) and PAH-albumin adducts (p = 0.002). The influence of the glutatione S-transferase (GST) M1 deletion on the correlation between the biomarkers was studied in the combined group. A significant negative correlation was only observed between bulky carcinogen-DNA adducts and PAH-albumin adducts (p = 0.02) and between DNA adduct and urinary mutagenic activity (p = 0.02) in the GSTM1 null group, but not in the workers who were homozygotes or heterozygotes for GSTM1. Our results indicate that some of the selected biomarkers can be used to distinguish between high and low exposure to environmental genotoxins. (+info)
Detection of benzo[a]pyrene diol epoxide-DNA adducts in embryos from smoking couples: evidence for transmission by spermatozoa.
Tobacco smoking is deleterious to reproduction. Benzo[a]pyrene (B[a]P) is a potent carcinogen in cigarette smoke. Its reactive metabolite induces DNA-adducts, which can cause mutations. We investigated whether B[a]P diol epoxide (BPDE) DNA adducts are detectable in preimplantation embryos in relation to parental smoking. A total of 17 couples were classified by their smoking habits: (i) both partners smoke; (ii) wife non-smoker, husband smokes; and (iii) both partners were non-smokers. Their 27 embryos were exposed to an anti-BPDE monoclonal antibody that recognizes BPDE-DNA adducts. Immunostaining was assessed in each embryo and an intensity score was calculated for embryos in each smoking group. The proportion of blastomeres which stained was higher for embryos of smokers than for non-smokers (0.723 versus 0.310). The mean intensity score was also higher for embryos of smokers (1.40+/-0.28) than for non-smokers (0.38+/-0.14; P = 0.015), but was similar for both types of smoking couples. The mean intensity score was positively correlated with the number of cigarettes smoked by fathers (P = 0.02). Increased mean immunostaining in embryos from smokers, relative to non-smokers, indicates a relationship with parental smoking. The similar levels of immunostaining in embryos from both types of smoking couples suggest that transmission of modified DNA is mainly through spermatozoa. We confirmed paternal transmission of modified DNA by detection of DNA adducts in spermatozoa of a smoker father and his embryo. (+info)
In vitro reactions of butadiene monoxide with single- and double-stranded DNA: characterization and quantitation of several purine and pyrimidine adducts.
We have previously shown that butadiene monoxide (BM), the primary metabolite of 1,3-butadiene, reacted with nucleosides to form alkylation products that exhibited different rates of formation and different stabilities under in vitro physiological conditions. In the present study, BM was reacted with single-stranded (ss) and double-stranded (ds) calf thymus DNA and the alkylation products were characterized after enzymatic hydrolysis of the DNA. The primary products were regioisomeric N-7-guanine adducts. N-3-(2-hydroxy-3-buten-1-yl)adenine and N-3-(1-hydroxy-3-buten-2-yl)adenine, which were depurinated from the DNA more rapidly than the N-7-guanine adducts, were also formed. In addition, N6-(2-hydroxy-3-buten-1-yl)deoxyadenosine and N6-(1-hydroxy-3-buten-2-yl)deoxyadenosine were detected and evidence was obtained that these adducts were formed by Dimroth rearrangement of the corresponding N-1-deoxyadenosine adducts, not while in the DNA, but following the release of the N-1-alkylated nucleosides by enzymatic hydrolysis. N-3-(2-hydroxy-3-buten-1-yl)deoxyuridine adducts, which were apparently formed subsequent to deamination reactions of the corresponding deoxycytidine adducts, were also detected and were stable in the DNA. Adduct formation was linearly dependent upon BM concentration (10-1000 mM), with adduct ratios being similar at the various BM concentrations. At a high BM concentration (750 mM), the adducts were formed in a linear fashion for up to 8 h in both ssDNA and dsDNA. However, the rates of formation of the N-3-deoxyuridine and N6-deoxyadenosine adducts increased 10- to 20-fold in ssDNA versus dsDNA, whereas the N-7-guanine adducts increased only slightly, presumably due to differences in hydrogen bonding in ssDNA versus dsDNA. These results may contribute to a better understanding of the molecular mechanisms of mutagenesis and carcinogenesis of both BM and its parent compound, 1,3-butadiene. (+info)
Idoxifene derivatives are less reactive to DNA than tamoxifen derivatives, both chemically and in human and rat liver cells.
The drug tamoxifen shows evidence of genotoxicity, and induces liver tumours in rats. Covalent DNA adducts have been detected in the liver of rats treated with tamoxifen, and these arise through metabolism at the alpha-position to give an ester which reacts with DNA. (E)-1-(4-iodophenyl)-2-phenyl-1-[4-(2-pyrrolidinoethoxy)phenyl]-but-1-en e (idoxifene) is an analogue of tamoxifen in which formation of DNA adducts is greatly reduced; we could not detect any adducts in the DNA of cultured rat hepatocytes treated with 10 microM idoxifene, after analysis by the 32P-post-labelling method. The metabolite (Z)-4-(4-iodophenyl)-4-[4-(2-pyrrolidinoethoxy)phenyl]-3-phenyl-3-but en-2-ol (alpha-hydroxyidoxifene) gave adducts in rat hepatocytes, but far fewer than the corresponding tamoxifen metabolite. In human hepatocytes, neither idoxifene nor tamoxifen induced detectable levels of DNA adducts. We prepared the alpha-acetoxy ester of idoxifene as a model for the ultimate reactive metabolite formed in rat liver. It was less reactive than alpha-acetoxytamoxifen, as might be expected on mechanistic grounds. It reacted with DNA in the same way, to give adducts which were probably N2-alkyldeoxyguanosines, but to a lower extent. All these results indicate that idoxifene is much less genotoxic than tamoxifen, and should therefore be a safer drug. (+info)