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The Journal of Biological Chemistry May 2020Platinum-based chemotherapies, including oxaliplatin, are a mainstay in the management of solid tumors and induce cell death by forming intrastrand dinucleotide DNA...
Platinum-based chemotherapies, including oxaliplatin, are a mainstay in the management of solid tumors and induce cell death by forming intrastrand dinucleotide DNA adducts. Despite their common use, they are highly toxic, and approximately half of cancer patients have tumors that are either intrinsically resistant or develop resistance. Previous studies suggest that this resistance is mediated by variations in DNA repair levels or net drug influx. Here, we aimed to better define the roles of nucleotide excision repair and DNA damage in platinum chemotherapy resistance by profiling DNA damage and repair efficiency in seven oxaliplatin-sensitive and three oxaliplatin-resistant colorectal cancer cell lines. We assayed DNA repair indirectly as toxicity and directly measured bulky adduct formation and removal from the genome by slot blot and repair capacity in an excision assay, and used excision repair sequencing (XR-seq) to map repair events genome-wide at single-nucleotide resolution. Using this combinatorial approach and proxies for oxaliplatin-DNA damage, we observed no significant differences in repair efficiency that could explain the relative sensitivities and chemotherapy resistances of these cell lines. In contrast, the levels of oxaliplatin-induced DNA damage were significantly lower in the resistant cells, indicating that decreased damage formation, rather than increased damage repair, is a major determinant of oxaliplatin resistance in these cell lines. XR-seq-based analysis of gene expression revealed up-regulation of membrane transport pathways in the resistant cells, and these pathways may contribute to resistance. In conclusion, additional research is needed to characterize the factors mitigating cellular DNA damage formation by platinum compounds.
Topics: Colorectal Neoplasms; DNA Adducts; DNA Damage; DNA Repair; DNA, Neoplasm; Drug Resistance, Neoplasm; HCT116 Cells; Humans; Oxaliplatin
PubMed: 32299912
DOI: 10.1074/jbc.RA120.013347 -
Molecules (Basel, Switzerland) Jan 2019Thermal stabilities of DNA duplexes containing Gua (), α- () or β-anomer of formamidopyrimidine-N7-9-hydroxy-aflatoxin B₁ () differ markedly (T: a < g < b ), but the...
Thermal stabilities of DNA duplexes containing Gua (), α- () or β-anomer of formamidopyrimidine-N7-9-hydroxy-aflatoxin B₁ () differ markedly (T: a < g < b ), but the underlying molecular origin of this experimentally observed phenomenon is yet to be identified and determined. Here, by employing explicit-solvent molecular dynamics simulations coupled with free-energy calculations using a combined linear-interaction-energy/linear-response-approximation approach, we explain the quantitative differences in T m in terms of three structural features (bulkiness, order, and compactness) and three energetical contributions (non-polar, electrostatic, and preorganized-electrostatic), and thus advance the current understanding of the relationships between structures, free energies, and thermal stabilities of DNA double helices.
Topics: Aflatoxin B1; DNA; DNA Adducts; Models, Molecular; Nucleic Acid Conformation; Pyrimidines; Solvents; Structure-Activity Relationship; Temperature; Thermodynamics
PubMed: 30609733
DOI: 10.3390/molecules24010150 -
Chemical Research in Toxicology Oct 2012Exposure to endogenous and exogenous chemicals can lead to the formation of structurally modified DNA bases (DNA adducts). If not repaired, these nucleobase lesions can... (Review)
Review
Exposure to endogenous and exogenous chemicals can lead to the formation of structurally modified DNA bases (DNA adducts). If not repaired, these nucleobase lesions can cause polymerase errors during DNA replication, leading to heritable mutations and potentially contributing to the development of cancer. Because of their critical role in cancer initiation, DNA adducts represent mechanism-based biomarkers of carcinogen exposure, and their quantitation is particularly useful for cancer risk assessment. DNA adducts are also valuable in mechanistic studies linking tumorigenic effects of environmental and industrial carcinogens to specific electrophilic species generated from their metabolism. While multiple experimental methodologies have been developed for DNA adduct analysis in biological samples, including immunoassay, HPLC, and ³²P-postlabeling, isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) generally has superior selectivity, sensitivity, accuracy, and reproducibility. As typical DNA adduct concentrations in biological samples are between 0.01-10 adducts per 10⁸ normal nucleotides, ultrasensitive HPLC-ESI-MS/MS methodologies are required for their analysis. Recent developments in analytical separations and biological mass spectrometry, especially nanoflow HPLC, nanospray ionization MS, chip-MS, and high resolution MS, have pushed the limits of analytical HPLC-ESI-MS/MS methodologies for DNA adducts, allowing researchers to accurately measure their concentrations in biological samples from patients treated with DNA alkylating drugs and in populations exposed to carcinogens from urban air, drinking water, cooked food, alcohol, and cigarette smoke.
Topics: Animals; Chromatography, High Pressure Liquid; DNA Adducts; Equipment Design; Humans; Indicator Dilution Techniques; Isotopes; Mass Spectrometry
PubMed: 22827593
DOI: 10.1021/tx3002548 -
Basic & Clinical Pharmacology &... Sep 2017Human beings are exposed to many reactive electrophiles, both formed endogenously and from exogenous exposures. Such compounds could react with cellular biomolecules and... (Review)
Review
Human beings are exposed to many reactive electrophiles, both formed endogenously and from exogenous exposures. Such compounds could react with cellular biomolecules and form stable reaction products, adducts, at nucleophilic sites in proteins and DNA, constituting a risk for toxic effects. Adductomic approaches aim to study the totality of adducts, to specific biomolecules, by mass spectrometric screening. This Mini-Review focuses on the development and application of an adductomic approach for the screening of unknown adducts to N-terminal valine (Val) in haemoglobin (Hb) by liquid chromatography tandem mass spectrometry (LC-MS/MS). The approach is based on the FIRE procedure, a modified Edman procedure for the analysis of adducts to N-terminal Val in Hb by LC-MS/MS. In the first application of the approach, samples from 12 smokers/non-smokers were screened for Hb adducts, and six previously identified adducts and 20 unknown adducts were detected. To confirm the observation of the detected unknown adducts, targeted screenings were performed in larger sets of blood samples (n = 50-120) from human cohorts. The majority of the previously detected unknown adducts was found in all analysed samples, with large interindividual variations in adduct levels. For structural identification of unknown adducts, a strategy using adductome LC-MS/MS data was formulated and applied. Six identified adducts correspond to ethylation and the precursor electrophiles ethyl vinyl ketone, glyoxal, methylglyoxal, acrylic acid and 1-octen-3-one. The observation of these adducts in human blood motivate further studies to evaluate possible contributions to health risks, as well as their potential as biomarkers of exposure.
Topics: Aldehydes; Biomarkers; Chromatography, Liquid; DNA Adducts; Environmental Exposure; Epoxy Compounds; Hemoglobins; Humans; Nitrosamines; Tandem Mass Spectrometry; Valine
PubMed: 27889941
DOI: 10.1111/bcpt.12715 -
International Journal of Molecular... Sep 2021The plant extract aristolochic acid (AA), containing aristolochic acids I (AAI) and II (AAII) as major components, causes aristolochic acid nephropathy (AAN) and Balkan...
The plant extract aristolochic acid (AA), containing aristolochic acids I (AAI) and II (AAII) as major components, causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN), unique renal diseases associated with upper urothelial cancer. Recently (Chemical Research in Toxicology 33(11), 2804-2818, 2020), we showed that the metabolism of AAI and AAII in Wistar rats is influenced by their co-exposure (i.e., AAI/AAII mixture). Using the same rat model, we investigated how exposure to the AAI/AAII mixture can influence AAI and AAII DNA adduct formation (i.e., AA-mediated genotoxicity). Using P-postlabelling, we found that AA-DNA adduct formation was increased in the livers and kidneys of rats treated with AAI/AAII mixture compared to rats treated with AAI or AAII alone. Measuring the activity of enzymes involved in AA metabolism, we showed that enhanced AA-DNA adduct formation might be caused partially by both decreased AAI detoxification as a result of hepatic CYP2C11 inhibition during treatment with AAI/AAII mixture and by hepatic or renal NQO1 induction, the key enzyme predominantly activating AA to DNA adducts. Moreover, our results indicate that AAII might act as an inhibitor of AAI detoxification . Consequently, higher amounts of AAI might remain in liver and kidney tissues, which can be reductively activated, resulting in enhanced AAI DNA adduct formation. Collectively, these results indicate that AAII present in the plant extract AA enhances the genotoxic properties of AAI (i.e., AAI DNA adduct formation). As patients suffering from AAN and BEN are always exposed to the plant extract (i.e., AAI/AAII mixture), our findings are crucial to better understanding host factors critical for AAN- and BEN-associated urothelial malignancy.
Topics: Animals; Aristolochic Acids; Carcinogenesis; Carcinogens; DNA Adducts; DNA, Neoplasm; Male; Rats; Rats, Wistar
PubMed: 34638820
DOI: 10.3390/ijms221910479 -
Mutation Research Dec 2006Aristolochic acid (AA) is a potent nephrotoxin and carcinogen and is the causative factor for Chinese herb nephropathy. AA has been associated with the development of... (Comparative Study)
Comparative Study
Aristolochic acid (AA) is a potent nephrotoxin and carcinogen and is the causative factor for Chinese herb nephropathy. AA has been associated with the development of urothelial cancer in humans, and kidney and forestomach tumors in rodents. To investigate the molecular mechanisms responsible for the tumorigenicity of AA, we determined the DNA adduct formation and mutagenicity of AA in the liver (nontarget tissue) and kidney (target tissue) of Big Blue rats. Groups of six male rats were gavaged with 0, 0.1, 1.0 and 10.0 mg AA/kg body weight five times/week for 3 months. The rats were sacrificed 1 day after the final treatment, and the livers and kidneys were isolated. DNA adduct formation was analyzed by 32P-postlabeling and mutant frequency (MF) was determined using the lambda Select-cII Mutation Detection System. Three major adducts (7-[deoxyadenosin-N6-yl]-aristolactam I, 7-[deoxyadenosin-N6-yl]-aristolactam II and 7-[deoxyguanosin-N2-yl]-aristolactam I) were identified. There were strong linear dose-responses for AA-induced DNA adducts in treated rats, ranging from 25 to 1967 adducts/10(8) nucleotides in liver and 95-4598 adducts/10(8) nucleotides in kidney. A similar trend of dose-responses for mutation induction also was found, the MFs ranging from 37 to 666 x 10(-6) in liver compared with the MFs of 78-1319 x 10(-6) that we previously reported for the kidneys of AA-treated rats. Overall, kidneys had at least two-fold higher levels of DNA adducts and MF than livers. Sequence analysis of the cII mutants revealed that there was a statistically significant difference between the mutation spectra in both kidney and liver of AA-treated and control rats, but there was no significant difference between the mutation spectra in AA-treated livers and kidneys. A:T-->T:A transversion was the predominant mutation in AA-treated rats; whereas G:C-->A:T transition was the main type of mutation in control rats. These results indicate that the AA treatment that eventually results in kidney tumors in rats also results in significant increases in DNA adduct formation and cII MF in kidney. Although the same treatment does not produce tumors in rat liver, it does induce DNA adducts and mutations in this tissue, albeit at lower levels than in kidney.
Topics: Animals; Aristolochic Acids; DNA Adducts; Kidney; Liver; Mutagens; Mutation; Rats; Rats, Inbred Strains
PubMed: 17010389
DOI: 10.1016/j.mrfmmm.2006.08.004 -
International Journal of Molecular... Dec 2022Organoids are 3D cultures that to some extent reproduce the structure, composition and function of the mammalian tissues from which they derive, thereby creating in...
Organoids are 3D cultures that to some extent reproduce the structure, composition and function of the mammalian tissues from which they derive, thereby creating in vitro systems with more in vivo-like characteristics than 2D monocultures. Here, the ability of human organoids derived from normal gastric, pancreas, liver, colon and kidney tissues to metabolise the environmental carcinogen benzo[]pyrene (BaP) was investigated. While organoids from the different tissues showed varied cytotoxic responses to BaP, with gastric and colon organoids being the most susceptible, the xenobiotic-metabolising enzyme (XME) genes, and , were highly upregulated in all organoid types, with kidney organoids having the highest levels. Furthermore, the presence of two key metabolites, BaP--7,8-dihydrodiol and BaP-tetrol-l-1, was detected in all organoid types, confirming their ability to metabolise BaP. BaP bioactivation was confirmed both by the activation of the DNA damage response pathway (induction of p-p53, pCHK2, p21 and γ-H2AX) and by DNA adduct formation. Overall, pancreatic and undifferentiated liver organoids formed the highest levels of DNA adducts. Colon organoids had the lowest responses in DNA adduct and metabolite formation, as well as XME expression. Additionally, high-throughput RT-qPCR explored differences in gene expression between organoid types after BaP treatment. The results demonstrate the potential usefulness of organoids for studying environmental carcinogenesis and genetic toxicology.
Topics: Humans; Activation, Metabolic; Benzo(a)pyrene; Cytochrome P-450 CYP1A1; DNA Adducts; Liver; Organoids
PubMed: 36614051
DOI: 10.3390/ijms24010606 -
Toxicological Sciences : An Official... Jan 2022Vinyl acetate monomer (VAM) is heavily used to synthesize polymers. Previous studies have shown that inhaled VAM, being metabolized to acetaldehyde, may form DNA adducts...
Vinyl acetate monomer (VAM) is heavily used to synthesize polymers. Previous studies have shown that inhaled VAM, being metabolized to acetaldehyde, may form DNA adducts including N2-ethylidene-deoxyguanosine (N2-EtD-dG), which may subsequently cause mutations and contribute to its carcinogenesis. Currently, there is little knowledge on the molecular dosimetry between VAM exposure and DNA adducts under dosages relevant to human exposure. In this study, 0.02, 0.1, 1, 10, 50, 200, and 600 ppm VAM were exposed to rats by inhalation for 14 days (6 h/day). The use of [13C2]-VAM allows unambiguous differentiation and quantification of the exogenous and endogenous N2-EtD-dG by highly sensitive LC-MS/MS. Our data indicate that VAM-induced exogenous DNA adducts were formed in a non-linear manner. Exogenous DNA adducts were only detected in the nasal epithelium of rats exposed to 10, 50, 200, and 600 ppm VAM, whereas endogenous adducts were found in all nasal and other tissues analyzed. In addition, ratios of exogenous/endogenous DNA adducts were less than 1 with the dose up to 50 ppm, indicating that endogenous DNA adducts are predominant at low VAM concentrations. Moreover, differential dose-response in terms of exogenous DNA adduct formation were observed between nasal respiratory and olfactory epithelium. Furthermore, the lack of exogenous DNA adducts in distant tissues, including peripheral blood mononuclear cells, liver, brain, and bone marrow, indicates that VAM and/or its metabolite do not distribute systemically to cause DNA damage in distant tissues. Together, these results provided new molecular dosimetry to improve science-based cancer risk assessments of VAM.
Topics: Animals; Chromatography, Liquid; DNA Adducts; Leukocytes, Mononuclear; Rats; Tandem Mass Spectrometry; Vinyl Compounds
PubMed: 34904679
DOI: 10.1093/toxsci/kfab140 -
International Journal of Molecular... Oct 2021DNA-dependent DNA and RNA polymerases are important modulators of biological functions such as replication, transcription, recombination, or repair. In this work...
Processing and Bypass of a Site-Specific DNA Adduct of the Cytotoxic Platinum-Acridinylthiourea Conjugate by Polymerases Involved in DNA Repair: Biochemical and Thermodynamic Aspects.
DNA-dependent DNA and RNA polymerases are important modulators of biological functions such as replication, transcription, recombination, or repair. In this work performed in cell-free media, we studied the ability of selected DNA polymerases to overcome a monofunctional adduct of the cytotoxic/antitumor platinum-acridinylthiourea conjugate [PtCl(en)(L)](NO) (en = ethane-1,2-diamine, L = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea) (ACR) in its favored 5'-CG sequence. We focused on how a single site-specific ACR adduct with intercalation potency affects the processivity and fidelity of DNA-dependent DNA polymerases involved in translesion synthesis (TLS) and repair. The ability of the G(N7) hybrid ACR adduct formed in the 5'-TCGT sequence of a 24-mer DNA template to inhibit the synthesis of a complementary DNA strand by the exonuclease-deficient Klenow fragment of DNA polymerase I (KF) and human polymerases eta, kappa, and iota was supplemented by thermodynamic analysis of the polymerization process. Thermodynamic parameters of a simulated translesion synthesis across the ACR adduct were obtained by using microscale thermophoresis (MST). Our results show a strong inhibitory effect of an ACR adduct on enzymatic TLS: there was only small synthesis of a full-length product (less than 10%) except polymerase eta (~20%). Polymerase eta was able to most efficiently bypass the ACR hybrid adduct. Incorporation of a correct dCMP opposite the modified G residue is preferred by all the four polymerases tested. On the other hand, the frequency of misinsertions increased. The relative efficiency of misinsertions is higher than that of matched cytidine monophosphate but still lower than for the nonmodified control duplex. Thermodynamic inspection of the simulated TLS revealed a significant stabilization of successively extended primer/template duplexes containing an ACR adduct. Moreover, no significant decrease of dissociation enthalpy change behind the position of the modification can contribute to the enzymatic TLS observed with the DNA-dependent, repair-involved polymerases. This TLS could lead to a higher tolerance of cancer cells to the ACR conjugate compared to its enhanced analog, where thiourea is replaced by an amidine group: [PtCl(en)(L)](NO) (complex AMD, en = ethane-1,2-diamine, L = N-[2-(acridin-9-ylamino)ethyl]-N-methylpropionamidine).
Topics: DNA Adducts; DNA Damage; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Humans; Intercalating Agents; Organoplatinum Compounds; Urea
PubMed: 34639179
DOI: 10.3390/ijms221910838 -
Chemical Research in Toxicology Jan 2018The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a powerful lung carcinogen in animal models and is considered a causative factor...
Methyl DNA Phosphate Adduct Formation in Rats Treated Chronically with 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and Enantiomers of Its Metabolite 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol.
The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a powerful lung carcinogen in animal models and is considered a causative factor for lung cancer in tobacco users. NNK is stereoselectively and reversibly metabolized to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which is also a lung carcinogen. Both NNK and NNAL undergo metabolic activation by α-hydroxylation on their methyl groups to form pyridyloxobutyl and pyridylhydroxybutyl DNA base and phosphate adducts, respectively. α-Hydroxylation also occurs on the α-methylene carbons of NNK and NNAL to produce methane diazohydroxide, which reacts with DNA to form methyl DNA base adducts. DNA adducts of NNK and NNAL are important in their mechanisms of carcinogenesis. In this study, we characterized and quantified methyl DNA phosphate adducts in the lung of rats treated with 5 ppm of NNK, (S)-NNAL, or (R)-NNAL in drinking water for 10, 30, 50, and 70 weeks, by using a novel liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method. A total of 23, 21, and 22 out of 32 possible methyl DNA phosphate adducts were detected in the lung tissues of rats treated with NNK, (S)-NNAL, and (R)-NNAL, respectively. Levels of the methyl DNA phosphate adducts were 2290-4510, 872-1120, and 763-1430 fmol/mg DNA, accounting for 15-38%, 8%, and 5-9% of the total measured DNA adducts in rats treated with NNK, (S)-NNAL, and (R)-NNAL, respectively. The methyl DNA phosphate adducts characterized in this study further enriched the diversity of DNA adducts formed by NNK and NNAL. These results provide important new data regarding NNK- and NNAL-derived DNA damage and new insights pertinent to future mechanistic and biomonitoring studies of NNK, NNAL, and other chemical methylating agents.
Topics: Animals; DNA Adducts; DNA Methylation; Hydrolysis; Lung; Male; Molecular Structure; Nitrosamines; Phosphates; Rats; Rats, Inbred F344; Stereoisomerism
PubMed: 29131934
DOI: 10.1021/acs.chemrestox.7b00281