Possible Genetic Risks from Heat-Damaged DNA in Food

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The consumption of foods prepared at high temperatures has been associated with numerous health risks. To date, the chief identified source of risk has been small molecules produced in trace levels by cooking and reacting with healthy DNA upon consumption. Here, we considered whether the DNA in food itself also presents a hazard. We hypothesize that high-temperature cooking may cause significant damage to the DNA in food, and this damage might find its way into cellular DNA by metabolic salvage. We tested cooked and raw foods and found high levels of hydrolytic and oxidative damage to all four DNA bases upon cooking. Exposing cultured cells to damaged 2′-deoxynucleosides (particularly pyrimidines) resulted in elevated DNA damage and repair responses in the cells. Feeding a deaminated 2′-deoxynucleoside (2′-deoxyuridine), and DNA containing it, to mice resulted in substantial uptake into intestinal genomic DNA and promoted double-strand chromosomal breaks there. The results suggest the possibility of a previously unrecognized pathway whereby high-temperature cooking may contribute to genetic risks.

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Synopsis

Cooking food at high temperature is found to damage DNA in foods. Incubating damaged DNA components with cells, or feeding them to mice, results in damage to genomic DNA.

Cooking foods at high temperatures has been associated with numerous health risks. (1) The consumption of red meat, which is frequently prepared at high temperature, is associated with colorectal and pancreatic cancer as well as metabolic syndromes such as type 2 diabetes and cardiovascular disease, and this consumption is also negatively associated with longevity. (2) High-temperature cooking of certain vegetables for consumption is also associated with disease risk. (3) Numerous mechanistic studies have implicated chemical changes in cooked food with damage caused to human DNA. (1,4) This has led the Food and Drug Administration (FDA) to recommend reductions in the public consumption of red meat and of deep-fried foods in general.

Studies aimed at delineating possible mechanisms of these pathologic associations have focused on small-molecule metabolites that can react with DNA. For example, polycyclic aromatic hydrocarbons (PAHs) and heterocyclic amines (HCAs) are produced at trace levels during the cooking of food and then bioactivated upon consumption into reactive species that alkylate DNA, resulting in the accumulation of damage and mutations over years of exposure (Figure 1). (5) Other reactive and potentially carcinogenic small molecules generated during high-temperature cooking include aldehydes, acrylamide, and N-nitroso compounds which can alkylate DNA bases. (1) When such species react with DNA, this can result in mutations when replication specificity is altered by modified nucleobases and in genotoxicity and chromosomal rearrangements when strand breaks occur during repair.

Our data represent, to our knowledge, the first documentation of damage to food DNA as a result of cooking and suggest a possible new etiology for genetic risk from cooked foods. Our findings add support to previous conclusions that high-temperature cooking confers a significant health risk with frequent and long-term consumption. (1,2) However, the new data suggests the possibility that a significant portion of the pathologic genetic dysfunction from cooked foods may plausibly arise from the consumption of food DNA itself, along with the previously identified small-molecule metabolites. As pointed out above, the consumption of cooked beef or pork in a meal can easily involve the ingestion of at least 1 g of DNA. (9) Our findings suggest that an estimated 6 × 10¹⁷ dU nucleotides (0.3 mg/1.0 μmol) and substantial quantities of other damaged 2′-deoxynucleotides may be ingested with 100 g of red meat mildly roasted for 20 min. This is as much as 3 to 4 orders of magnitude higher than the amounts of activated small-molecule metabolites such as HCAs and PAHs that occur in cooked food; (19) moreover, the damage resulting from salvaged 2′-deoxynucleosides (if incorporated via polymerases) is direct and requires no chance reaction with DNA. We do not discount the genetic risks that reactive small molecules in foods pose; indeed, these two mechanisms are not mutually exclusive.

Clearly, this initial study is very early, and establishing this hypothesized connection firmly will require more follow-up studies in toxicology. In addition, although our mechanistic hypothesis of salvaging damaged nucleosides is supported by several lines of evidence here (particularly for dU), we cannot yet rule out some unforeseen indirect mechanism whereby exposure to the damaged monomers found in cooked food elevates cellular DNA damage and subsequent repair responses.

It is likely that different cooking methods and diverse foods will result in large variations in DNA damage in the food. Our experiments revealed distinct differences in the level of damage by types of cooking, with roasting (220 °C) causing more damage than boiling (100 °C) relative to raw foods. Extended times at elevated temperatures have an important effect, as shown by our studies of DNA incubated at 95 °C over time. We note that our roasting procedure was relatively mild, and higher-temperature cooking methods (grilling, frying) and long times (smoking) are common in public use. More work is needed to test the effects of varied cooking procedures.

In the current studies, DNA from potatoes was substantially less damaged than was that from meats; the reason for this is not yet clear, although we speculate that the presence of high levels of starch may contribute to some protection against reactive oxygen species, perhaps by scavenging free radicals. (33) It remains to be seen if this holds true for other plant foods. Also potentially relevant is the fact that most plants are known to contain far smaller amounts of DNA per weight compared to animals (Supporting Information, Table 1). (9) The observation that plant-based diets (34) are association with lower cancer risks would also be consistent with these findings; further studies are required to better understand DNA damage in cooked plant-based foods relative to meats.

Many forms of damage are observed directly in cellular DNA, and cells have evolved numerous repair enzymes and pathways to address them. However, cells also present a line of defense against DNA damage even before it occurs in DNA, in the form of nucleotide pool sanitation enzymes. (35) We have observed that dU and 8-oxo-dG were the two most abundant forms of DNA damage in food emerging during cooking processes, and cells possess nucleotide sanitation enzymes (e.g., dUTPase and MTH1) (15) to specifically address deaminated and oxidized 2′-deoxynucleoside triphosphates. Prior studies have cited these enzymes’ function to defend against spontaneous deamination and oxidation that arise intracellularly during normal metabolism. We suggest the additional possibility that their activities are also crucial to defending against the consumption and salvage of damaged DNA components from food. Indeed, we show that the suppression of dUTPase activity markedly increases levels of DNA damage in the presence of dU in the medium (Figure 3e). Certain human populations are known to possess genetically attenuated nucleotide sanitization activities or DNA repair activities, (36) and the new findings, if confirmed more broadly, suggest that high-temperature cooking may pose yet more serious risks to these individuals, especially with frequent consumption over years. Future population studies will be helpful in establishing such a connection.

Taken together, our experiments suggest a possible novel mechanism that has the potential to help explain connections between high-temperature cooking (particularly of meats) and human cancers and metabolic diseases. The results prompt the need for further studies to assess the effects of long-term exposure at lower concentrations to determine which specific damaged DNA species are of greatest concern. If additional studies support these early findings, then this suggests new reasons to emphasize food preparation at reduced temperatures and times as well as the consumption of vegetables and raw foods in general.

Finally, we note that, in addition to possible relevance to diet, the observation of the salvaging of damaged nucleosides into cells and tissues may serve as a useful tool in future studies of DNA damage and repair. Typically, researchers employ general mechanisms (such as adding oxidizing species to the medium) to induce cellular DNA damage, resulting in the formation of several species simultaneously. The proposed nucleoside salvage mechanism suggests the possibility of introducing specific damaged species one at a time into cells; future work will explore this possibility.

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    Hizi, A.; Herzig, E. dUTPase: the Frequently Overlooked Enzyme Encoded by Many Retroviruses. Retrovirology 2015, 12, 70,  DOI: 10.1186/s12977-015-0198-9

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    dUTPase: the frequently overlooked enzyme encoded by many retroviruses

    Hizi Amnon; Herzig Eytan

    Retrovirology (2015), 12 (), 70 ISSN:.

    Retroviruses are among the best studied viruses in last decades due to their pivotal involvement in cellular processes and, most importantly, in causing human diseases, most notably-acquired immunodeficiency syndrome (AIDS) that is triggered by human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2, respectively). Numerous studied were conducted to understand the involvement of the three cardinal retroviral enzymes, reverse transcriptase, integrase and protease, in the life cycle of the viruses. These studies have led to the development of many inhibitors of these enzymes as anti-retroviral specific drugs that are used for routine treatments of HIV/AIDS patients. Interestingly, a fourth virus-encoded enzyme, the deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is also found in several major retroviral groups. The presence and the importance of this enzyme to the life cycle of retroviruses were usually overlooked by most retrovirologists, although the occurrence of dUTPases, particularly in beta-retroviruses and in non-primate retroviruses, is known for more than 20 years. Only more recently, retroviral dUTPases were brought into the limelight and were shown in several cases to be essential for viral replication. Therefore, it is likely that future studies on this enzyme will advance our knowledge to a level that will allow designing novel, specific and potent anti-dUTPase drugs that are effective in combating retroviral diseases. The aim of this review is to give concise background information on dUTPases in general and to summarize the most relevant data on retroviral dUTPases and their involvement in the replication processes and pathogenicity of the viruses, as well as in possibly-associated human diseases.

    Gad, H.; Koolmeister, T.; Jemth, A.-S.; Eshtad, S.; Jacques, S. A.; Ström, C. E.; Svensson, L. M.; Schultz, N.; Lundbäck, T.; Einarsdottir, B. O. MTH1 Inhibition Eradicates Cancer by Preventing Sanitation of the dNTP Pool. Nature 2014, 508, 215– 221,  DOI: 10.1038/nature13181

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    MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool

    Gad, Helge; Koolmeister, Tobias; Jemth, Ann-Sofie; Eshtad, Saeed; Jacques, Sylvain A.; Stroem, Cecilia E.; Svensson, Linda M.; Schultz, Niklas; Lundbaeck, Thomas; Einarsdottir, Berglind Osk; Saleh, Aljona; Gokturk, Camilla; Baranczewski, Pawel; Svensson, Richard; Berntsson, Ronnie P.-A.; Gustafsson, Robert; Stroemberg, Kia; Sanjiv, Kumar; Jacques-Cordonnier, Marie-Caroline; Desroses, Matthieu; Gustavsson, Anna-Lena; Olofsson, Roger; Johansson, Fredrik; Homan, Evert J.; Loseva, Olga; Braeutigam, Lars; Johansson, Lars; Hoeglund, Andreas; Hagenkort, Anna; Pham, Therese; Altun, Mikael; Gaugaz, Fabienne Z.; Vikingsson, Svante; Evers, Bastiaan; Henriksson, Martin; Vallin, Karl S. A.; Wallner, Olov A.; Hammarstroem, Lars G. J.; Wiita, Elisee; Almloef, Ingrid; Kalderen, Christina; Axelsson, Hanna; Djureinovic, Tatjana; Puigvert, Jordi Carreras; Haeggblad, Maria; Jeppsson, Fredrik; Martens, Ulf; Lundin, Cecilia; Lundgren, Bo; Granelli, Ingrid; Jensen, Annika Jenmalm; Artursson, Per; Nilsson, Jonas A.; Stenmark, Pal; Scobie, Martin; Berglund, Ulrika Warpman; Helleday, Thomas

    Nature (London, United Kingdom) (2014), 508 (7495), 215-221CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)

    Cancers have dysfunctional redox regulation resulting in reactive oxygen species prodn., damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, the authors show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. The authors validate MTH1 as an anticancer target in vivo and describe small mols. TH287 (I) and TH588 (II) as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.

16. 16

    Wilson, D. L.; Kool, E. T. Ultrafast Oxime Formation Enables Efficient Fluorescence Light-Up Measurement of DNA Base Excision. J. Am. Chem. Soc. 2019, 141, 19379– 19388,  DOI: 10.1021/jacs.9b09812

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    Ultrafast Oxime Formation Enables Efficient Fluorescence Light-up Measurement of DNA Base Excision

    Wilson, David L.; Kool, Eric T.

    Journal of the American Chemical Society (2019), 141 (49), 19379-19388CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)

    DNA glycosylases constitute a biol. and biomedically important group of DNA repair enzymes responsible for initiating base excision repair (BER). Measuring their activities can be useful for studying the mechanisms DNA damage and repair and for practical applications in cancer diagnosis and drug screening. Previous fluorescence methods for assaying DNA glycosylases are often complex and/or limited in scope to a single enzyme type. Here we report a universal base excision reporter (UBER) fluorescence probe design that implements an unprecedentedly rapid oxime reaction (>150 M-1 s-1) with high specificity for the abasic (AP) site of DNA. The mol. rotor design achieves a robust >250-500-fold increase in fluorescence upon reaction with AP sites in DNA. By using the fluorescence reporter in concert with specific DNA lesion-contg. substrates, the UBER probe can be used in a coupled assay in principle with any DNA glycosylase. We demonstrate the utility of the UBER probe by assaying five different glycosylases in real time as well as profiling glycosylase activity in cell lysates. We anticipate that the UBER probe will be of considerable utility to researchers studying DNA repair biol. owing to its high level of generalizability, ease of use, and compatibility with biol. derived samples.

17. 17

    Herbel, W.; Montag, A. Nucleo-Compounds in Protein-Rich Food. Unters. Forsch. 1987, 185, 119– 122,  DOI: 10.1007/BF01850090

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    Nucleo-compounds in protein rich food

    Herbel, Walter; Montag, Alfred

    Zeitschrift fuer Lebensmittel-Untersuchung und -Forschung (1987), 185 (2), 119-22CODEN: ZLUFAR; ISSN:0044-3026.

    RNA, DNA, IMP, and pyrimidine and purine bases were detd. in several protein-high foods and the results were tabulated. Heat treatment of the food caused considerable changes in the contents of these substances.

    Lassek, E.; Montag, A. Nucleic Acid Components in Carbohydrate-Rich Food. Unters. Forsch. 1990, 190, 17– 21,  DOI: 10.1007/BF01188257

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    Nucleic acid components in carbohydrate-rich food

    Lassek, Eva; Montag, Alfred

    Zeitschrift fuer Lebensmittel-Untersuchung und -Forschung (1990), 190 (1), 17-21CODEN: ZLUFAR; ISSN:0044-3026.

    The nucleic acid component content of numerous foods, esp. those rich in carbohydrates, was investigated. The data obtained for bases (purines and pyrimidines) were calcd. as nucleic acid equiv. (RNA or DNA); the inosine monophosphate content was calcd. from the measured content of hypoxanthine. Not only did cultivated plants such as cereals and pulses show a high RNA-equiv. content but also vegetables such as spinach, leek, broccoli, Chinese cabbage, and cauliflower and mushrooms, including oyster, flat, button and cap mushrooms. In many vegetarian instant meals (principally soups, the presence of autolyzed or hydrolyzed yeast resulted in high purine contents. Most natural foods contg. resting cell tissue, such as grains of seed, exhibit only high-mol.-mass components of various concns.; growing cell tissues (e.g. soybean sprouts) show, however, some low-mol.-mass compds. in addn. to these.

18. 18

    Lewis, C. A.; Crayle, J.; Zhou, S.; Swanstrom, R.; Wolfenden, R. Cytosine Deamination and the Precipitous Decline of Spontaneous Mutation during Earth’s History. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 8194– 8199,  DOI: 10.1073/pnas.1607580113

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    18

    Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history

    Lewis, Charles A. Jr.; Crayle, Jesse; Zhou, Shuntai; Swanstrom, Ronald; Wolfenden, Richard

    Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (29), 8194-8199CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)

    The hydrolytic deamination of cytosine and 5-methylcytosine residues in DNA appears to contribute significantly to the appearance of spontaneous mutations in microorganisms and in human disease. Here, the authors examd. the mechanism of cytosine deamination and the response of the uncatalyzed reaction to changing temp. The pos. charged 1,3-dimethylcytosinium ion was hydrolyzed at a rate similar to the rate of acid-catalyzed hydrolysis of 1-methylcytosine, for which it furnished a satisfactory kinetic model and a probable mechanism. In agreement with earlier reports, uncatalyzed deamination was found to proceed at very similar rates for cytosine, 1-methylcytosine, cytidine, and cytidine 5'-phosphate, and also for cytosine residues in single-stranded DNA generated from a phagemid, in which the authors sequenced an insert representing the gene of HIV-1 virus protease. Arrhenius plots for the uncatalyzed deamination of cytosine were linear over the temp. range of 90-200° and indicated a heat of activation (ΔH⧧) of 23.4 ± 0.5 kcal/mol at pH 7. Recent evidence indicated that the surface of the Earth has been cool enough to support life for >4 × 109 yr and that life has been present for almost as long. If the temp. at Earth's surface is assumed to have followed Newton's law of cooling, declining exponentially from 100 to 25° during that period, then half of the cytosine-deaminating events per unit biomass would have taken place during the 0.2 × 109 yr, and <99.4% would have occurred during the 1st 2 × 109 yr.

19. 19

    Sahin, S.; Ulusoy, H. I.; Alemdar, S.; Erdogan, S.; Agaoglu, S. The Presence of Polycyclic Aromatic Hydrocarbons (PAHs) in Grilled Beef, Chicken and Fish by Considering Dietary Exposure and Risk Assessment. Food Sci. Anim. Resour. 2020, 40, 675– 688,  DOI: 10.5851/kosfa.2020.e43

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    The Presence of Polycyclic Aromatic Hydrocarbons (PAHs) in Grilled Beef, Chicken and Fish by Considering Dietary Exposure and Risk Assessment

    Sahin Seyda; Alemdar Suleyman; Agaoglu Sema; Ulusoy Halil Ibrahim; Erdogan Selim

    Food science of animal resources (2020), 40 (5), 675-688 ISSN:.

    Polycyclic aromatic hydrocarbons (PAHs) are dangerous chemical compounds that can be formed by cooking foods at high temperatures. The aim of this study is to determine the level of contamination of PAH compounds with high performance liquid chromatography (HPLC) on heat treated meat samples and the consumption of PAH compounds in meat samples, as well as the dietary exposure status and possible health risk estimation. In five different heat treated meat samples (meat doner, chicken doner, meatballs, grilled chicken, and fish), the total PAH (Σ16PAH) contamination level was 6.08, 4.42, 4.45, 4.91, and 7.26 μg/kg, respectively. Benzo[a]pyrene (BaP) in meatballs and grilled fish samples had a level 0.70 and 0.73 μg/kg. All of the samples analyzed were found to be below the EU permitted limit (5 μg/kg) in terms of BaP. Estimates of daily intake (EDI) for a total of 16PAH in heat treated meat doner, chicken doner, meatballs, grilled chicken and fish samples were 3.41, 3.71, 2.49, 4.12, and 1.77 ng/kg bw/day, respectively. In this study, the average margin of exposure (MOE) value calculated was found in the range of 179.487 and 425.000 for BaP and PAH4. This study is the first study to provide important information in terms of evaluating the possible health risk that PAH compounds can create in people's diets due to heat treatment of meat and meat products in Sivas, Turkey.

    Sinha, R.; Rothman, N.; Salmon, C.; Knize, M.; Brown, E.; Swanson, C.; Rhodes, D.; Rossi, S.; Felton, J.; Levander, O. Heterocyclic Amine Content in Beef Cooked by Different Methods to Varying Degrees of Doneness and Gravy Made from Meat Drippings. Food Chem. Toxicol. 1998, 36, 279– 287,  DOI: 10.1016/S0278-6915(97)00162-2

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    19

    Heterocyclic amine content in beef cooked by different methods to varying degrees of doneness and gravy made from meat drippings

    Sinha, R.; Rothman, N.; Salmon, C. P.; Knize, M. G.; Brown, E. D.; Swanson, C. A.; Rhodes, D.; Rossi, S.; Felton, J. S.; Levander, O. A.

    Food and Chemical Toxicology (1998), 36 (4), 279-287CODEN: FCTOD7; ISSN:0278-6915. (Elsevier Science Ltd.)

    Meats cooked at high temps. sometimes contain heterocyclic amines (HCAs) that are known mutagens and animal carcinogens, but their carcinogenic potential in humans has not been established. To investigate the assocn. between HCAs and cancer, sources of exposure to these compds. need to be detd. Beef is the most frequently consumed meat in the United States and for this study we detd. HCA values in beef samples cooked in ways to represent US cooking practices, the results of which can be used in epidemiol. studies to est. HCA exposure from dietary questionnaires. We measured five HCAs [2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP)] in different types of cooked beef using solid-phase extn. and HPLC. Steak and hamburger patties were pan-fried, oven-broiled, and grilled/barbecued to four levels of doneness (rare, medium, well done or very well done), while beef roasts were oven cooked to three levels of doneness (rare, medium or well done). The measured values of the specific HCAs varied with the cut of beef, cooking method, and doneness level. In general, MeIQx content increased with doneness under each cooking condition for steak and hamburger patties, up to 8.2 ng/g. PhIP was the predominant HCA produced in steak (1.9 to 30 ng/g), but was formed only in very well done fried or grilled hamburger. DiMeIQx was found in trace levels in pan-fried steaks only, while IQ and MeIQ were not detectable in any of the samples. Roast beef did not contain any of the HCAs, but the gravy made from the drippings from well done roasts had 2 ng/g of PhIP and 7 ng/g of MeIQx. Epidemiol. studies need to consider the type of meat, cooking method and degree of doneness/surface browning in survey questions to adequately assess an individual's exposure to HCAs.

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    Chatgilialoglu, C.; Ferreri, C.; Geacintov, N. E.; Krokidis, M. G.; Liu, Y.; Masi, A.; Shafirovich, V.; Terzidis, M. A.; Tsegay, P. S. 5′, 8-Cyclopurine Lesions in DNA Damage: Chemical, Analytical, Biological, and Diagnostic Significance. Cells 2019, 8, 513,  DOI: 10.3390/cells8060513

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    5',8-cyclopurine lesions in DNA damage: chemical, analytical, biological, and diagnostic significance

    Chatgilialoglu, Chryssostomos; Ferreri, Carla; Geacintov, Nicholas E.; Krokidis, Marios G.; Liu, Yuan; Masi, Annalisa; Shafirovich, Vladimir; Terzidis, Michael A.; Tsegay, Pawlos S.

    Cells (2019), 8 (6), 513CODEN: CELLC6; ISSN:2073-4409. (MDPI AG)

    Purine 5',8-cyclo-2'-deoxynucleosides (cPu) are tandem-type lesions obsd. among the DNA purine modifications and identified in mammalian cellular DNA in vivo. These lesions can be present in two diasteroisomeric forms, 5'R and 5'S, for each 2'-deoxyadenosine and 2'-deoxyguanosine moiety. They are generated exclusively by hydroxyl radical attack to 2'-deoxyribose units generating C5' radicals, followed by cyclization with the C8 position of the purine base. This review describes the main recent achievements in the prepn. of the cPu mol. library for anal. and DNA synthesis applications for the studies of the enzymic recognition and repair mechanisms, their impact on transcription and genetic instability, quant. detn. of the levels of lesions in various types of cells and animal model systems, and relationships between the levels of lesions and human health, disease, and aging, as well as the defining of the detection limits and quantification protocols.

21. 21

    Zauri, M.; Berridge, G.; Thézénas, M.-L.; Pugh, K. M.; Goldin, R.; Kessler, B. M.; Kriaucionis, S. CDA Directs Metabolism of Epigenetic Nucleosides Revealing a Therapeutic Window in Cancer. Nature 2015, 524, 114– 118,  DOI: 10.1038/nature14948

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    CDA directs metabolism of epigenetic nucleosides revealing a therapeutic window in cancer

    Zauri, Melania; Berridge, Georgina; Thezenas, Marie-Laetitia; Pugh, Kathryn M.; Goldin, Robert; Kessler, Benedikt M.; Kriaucionis, Skirmantas

    Nature (London, United Kingdom) (2015), 524 (7563), 114-118CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)

    Cells require nucleotides to support DNA replication and repair damaged DNA. In addn. to de novo synthesis, cells recycle nucleotides from the DNA of dying cells or from cellular material ingested through the diet. Salvaged nucleosides come with the complication that they can contain epigenetic modifications. Because epigenetic inheritance of DNA methylation mainly relies on copying of the modification pattern from parental strands, random incorporation of pre-modified bases during replication could have profound implications for epigenome fidelity and yield adverse cellular phenotypes. Although the salvage mechanism of 5-methyl-2'deoxycytidine (5mdC) has been investigated before, it remains unknown how cells deal with the recently identified oxidized forms of 5mdC: 5-hydroxymethyl-2'deoxycytidine (5hmdC), 5-formy-2'deoxycytidine (5fdC) and 5-carboxyl-2'deoxycytidine (5cadC). Here we show that enzymes of the nucleotide salvage pathway display substrate selectivity, effectively protecting newly synthesized DNA from the incorporation of epigenetically modified forms of cytosine. Thus, cell lines and animals can tolerate high doses of these modified cytidines without any deleterious effects on physiol. Notably, by screening cancer cell lines for growth defects after exposure to 5hmdC, we unexpectedly identify a subset of cell lines in which 5hmdC or 5fdC administration leads to cell lethality. Using genomic approaches, we show that the susceptible cell lines overexpress cytidine deaminase (CDA). CDA converts 5hmdC and 5fdC into variants of uridine that are incorporated into DNA, resulting in accumulation of DNA damage, and ultimately, cell death. Our observations extend current knowledge of the nucleotide salvage pathway by revealing the metab. of oxidized epigenetic bases, and suggest a new therapeutic option for cancers, such as pancreatic cancer, that have CDA overexpression and are resistant to treatment with other cytidine analogs.

    Gratzner, H. G. Monoclonal Antibody to 5-Bromo-and 5-Iododeoxyuridine: A New Reagent for Detection of DNA Replication. Science 1982, 218, 474– 475,  DOI: 10.1126/science.7123245

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    Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: a new reagent for detection of DNA replication

    Gratzner, Howard G.

    Science (Washington, DC, United States) (1982), 218 (4571), 474-5CODEN: SCIEAS; ISSN:0036-8075.

    Monoclonal antibodies specific for 5-bromodeoxyuridine have been produced and applied in detecting low levels of DNA replication on a cell-by-cell basis in vitro. The Ig-producing hybridomas were derived from spleen cells of mice immunized with a conjugate of iodouridine and ovalbumin. The cells were fused with the plasmacytoma line SP2/OAg14. The antibodies produced are highly specific for bromodeoxyuridine and iododeoxyuridine and do not crossreact with thymidine. DNA synthesis in cultured cells exposed to bromodeoxyuridine for as short a time as 6 min can be detected easily and rapidly by an immunofluorescent staining method and quantitated by flow cytometry.

    Salic, A.; Mitchison, T. J. A Chemical Method for Fast and Sensitive Detection of DNA Synthesis in vivo. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 2415– 2420,  DOI: 10.1073/pnas.0712168105

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    21

    A chemical method for fast and sensitive detection of DNA synthesis in vivo

    Salic, Adrian; Mitchison, Timothy J.

    Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (7), 2415-2420CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)

    We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) and its subsequent detection by a fluorescent azide through a Cu(I)-catalyzed [3 + 2] cycloaddn. reaction ("click" chem.). Detection of the EdU label is highly sensitive and can be accomplished in minutes. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration, allowing the staining of whole-mount prepns. of large tissue and organ explants. In contrast to BrdU, the method does not require sample fixation or DNA denaturation and permits good structural preservation. We demonstrate the use of the method in cultured cells and in the intestine and brain of whole animals.

22. 22

    Jun, Y. W.; Albarran, E.; Wilson, D. L.; Ding, J.; Kool, E. T. Fluorescence Imaging of Mitochondrial DNA Base Excision Repair Reveals Dynamics of Oxidative Stress Responses. Angew. Chem., Int. Ed. 2022, 61, e202111829,  DOI: 10.1002/anie.202111829

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    22

    Fluorescence Imaging of Mitochondrial DNA Base Excision Repair Reveals Dynamics of Oxidative Stress Responses

    Jun, Yong Woong; Albarran, Eddy; Wilson, David L.; Ding, Jun; Kool, Eric T.

    Angewandte Chemie, International Edition (2022), 61 (6), e202111829CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)

    Mitochondrial function in cells declines with aging and with neurodegeneration, due in large part to accumulated mutations in mitochondrial DNA (mtDNA) that arise from deficient DNA repair. However, measuring this repair activity is challenging. We employ a mol. approach for visualizing mitochondrial base excision repair (BER) activity in situ by use of a fluorescent probe (UBER) that reacts rapidly with AP sites resulting from BER activity. Administering the probe to cultured cells revealed signals that were localized to mitochondria, enabling selective observation of mtDNA BER intermediates. The probe showed elevated DNA repair activity under oxidative stress, and responded to suppression of glycosylase activity. Furthermore, the probe illuminated the time lag between the initiation of oxidative stress and the initial step of BER. Absence of MTH1 in cells resulted in elevated demand for BER activity upon extended oxidative stress, while the absence of OGG1 activity limited glycosylation capacity.

23. 23

    Bonda, E.; Rahav, G.; Kaya, A.; Bakhanashvili, M. p53 in the Mitochondria, as a Trans-Acting Protein, Provides Error-Correction Activities during the Incorporation of Non-Canonical dUTP into DNA. Oncotarget 2016, 7, 73323,  DOI: 10.18632/oncotarget.12331

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    p53 in the mitochondria, as a trans-acting protein, provides error-correction activities during the incorporation of non-canonical dUTP into DNA

    Bonda Elad; Rahav Galia; Kaya Angelina; Bakhanashvili Mary; Bakhanashvili Mary

    Oncotarget (2016), 7 (45), 73323-73336 ISSN:.

    Mutations in mitochondrial DNA is an outcome of errors produced by DNA polymerase γ during replication and failure of the repair mechanism. Misincorporation of non-canonical dUTP leads to mutagenesis or apoptosis, and may contribute to the cytotoxic effects of 5'-fluorouracil chemotherapy. Tumor suppressor p53 protein in the mitochondria displays physical and functional interactions with mitochondrial DNA and polymerase γ, and by its intrinsic 3'→5' exonuclease activity can diminish the polymerization errors. Here we demonstrate the impact of p53 on incorporation of uracil into DNA examined with mitochondrial fractions, as the source of polymerase γ. p53 in mitochondria facilitates DNA damage repair functions resulting from uracil-DNA misincorporation. Our biochemical studies revealed that the procession of U:A and mismatched U:G lesions enhances in the presence of recombinant or endogenous cytoplasmic p53. p53 in mitochondria can function as an exonuclease/proofreader for polymerase γ by either decreasing the incorporation of non-canonical dUTP into DNA or by promoting the excision of incorporated nucleotide from nascent DNA, thus expanding the spectrum of DNA damage sites exploited for proofreading as a trans-acting protein. The data suggest that p53 may contribute to defense of the cells from consequences of dUTP misincorporation in both normal and tumor cells.

24. 24

    Yano, W.; Yokogawa, T.; Wakasa, T.; Yamamura, K.; Fujioka, A.; Yoshisue, K.; Matsushima, E.; Miyahara, S.; Miyakoshi, H.; Taguchi, J. TAS-114, a First-in-Class Dual dUTPase/DPD Inhibitor, Demonstrates Potential to Improve Therapeutic Efficacy of Fluoropyrimidine-Based Chemotherapy. Mol. Cancer Ther. 2018, 17, 1683– 1693,  DOI: 10.1158/1535-7163.MCT-17-0911

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    24

    TAS-114, a First-in-Class Dual dUTPase/DPD Inhibitor, Demonstrates Potential to Improve Therapeutic Efficacy of Fluoropyrimidine-Based Chemotherapy

    Yano, Wakako; Yokogawa, Tatsushi; Wakasa, Takeshi; Yamamura, Keisuke; Fujioka, Akio; Yoshisue, Kunihiro; Matsushima, Eiji; Miyahara, Seiji; Miyakoshi, Hitoshi; Taguchi, Junko; Chong, Khoon Tee; Takao, Yayoi; Fukuoka, Masayoshi; Matsuo, Kenichi

    Molecular Cancer Therapeutics (2018), 17 (8), 1683-1693CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)

    In this study, we designed a novel small mol. inhibitor, TAS-114, which targets the intercellular metab. of 5-FU to enhance antitumor activity and modulates catabolic pathway to improve the systemic availability of 5-FU. TAS-114 strongly and competitively inhibited deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), a gatekeeper protein preventing aberrant base incorporation into DNA, and enhanced the cytotoxicity of fluoropyrimidines in cancer cells; however, it had little intrinsic activity. In addn., TAS-114 had moderate and reversible inhibitory activity on dihydropyrimidine dehydrogenase (DPD), a catabolizing enzyme of 5-FU. Thus, TAS-114 increased the bioavailability of 5-FU when coadministered with capecitabine in mice, and it significantly improved the therapeutic efficacy of capecitabine by reducing the required dose of the prodrug by dual enzyme inhibition. Enhancement of antitumor efficacy caused by the addn. of TAS-114 was retained in the presence of a potent DPD inhibitor contg. oral fluoropyrimidine (S-1), indicating that dUTPase inhibition plays a major role in enhancing the antitumor efficacy of fluoropyrimidine-based therapy. In conclusion, TAS-114, a dual dUTPase/DPD inhibitor, demonstrated the potential to improve the therapeutic efficacy of fluoropyrimidine. Dual inhibition of dUTPase and DPD is a novel strategy for the advancement of oral fluoropyrimidine-based chemotherapy for cancer treatment.

25. 25

    Ladner, R. D. The Role of dUTPase and Uracil-DNA Repair in Cancer Chemotherapy. Curr. Protein Pept. Sci. 2001, 2, 361– 370,  DOI: 10.2174/1389203013380991

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    The role of dUTPase and uracil-DNA repair in cancer chemotherapy

    Ladner, Robert D.

    Current Protein and Peptide Science (2001), 2 (4), 361-370CODEN: CPPSCM; ISSN:1389-2037. (Bentham Science Publishers Ltd.)

    A review with refs. Thymidylate metab. is an important target for chemotherapeutic agents that combat a variety of neoplastic diseases including head and neck, breast and gastrointestinal cancers. Therapeutic strategies applied to this pathway target the thymidylate synthase (TS) reaction that catalyzes the reductive methylation of deoxyuridylate (dUMP) to form thymidylate (TMP). This reaction represents the sole de novo source of TMP required for DNA replication and repair. Inhibitors of this pathway include the widely utilized fluoropyrimide and antifolate classes of anti-cancer agents. Studies attempting to elucidate the mol. mechanisms of cell killing mediated by inhibitors of the TS reaction suggest that cytotoxicity results from a process known as "thymineless death". This term describes the extreme TTP pool depletion obsd. following TS inhibition. Although depletion of TTP pools is clearly involved in this process, there is now considerable evidence implicating aberrant uracil-DNA metab. as an important mechanism of toxicity. Upon TS inhibition, dUTP pools may accumulate, inducing repeated cycles of uracil misincorporation into DNA and repair-mediated DNA damage. Central to the uracil-misincorporation pathway are the enzymes deoxyuridine nucleotidohydrolase (dUTPase) (EC 3.6.1.23) and uracil-DNA glycosylase (UDG) (EC 3.2.2.3). DUTPase catalyzes the hydrolysis of dUTP to form dUMP and pyrophosphate thereby eliminating dUTP and preventing its utilization by DNA polymerases during replication and repair. UDG initiates the base excision repair pathway effectively removing any uracil residues that may arise in DNA. Under normal conditions, uracil is precluded from DNA by the combined actions of dUTPase and UDG. However, during TS inhibition, dUTP pools may accumulate and overwhelm dUTPase, resulting in repeated cycles of uracil misincorporation and detrimental repair leading to stand breaks and cell death. Because dUTPase plays a pivotal role in regulating cellular dUTP pools, this enzyme could have profound effects on the efficacy of agents that target thymidylate biosynthesis. This article reviews our current understanding of the role of aberrant uracil-DNA metab. as a contributing mechanism of cytotoxicity initiated by chemotherapeutic agents that target de novo thymidylate metab. The role of dUTPase expression in modulating therapeutic response is presented including evidence from yeast and mammalian cell culture models and clin. studies. The regulation of human dUTPase isoforms in normal and neoplastic tissues will be reviewed as well as the role of dUTPase expression as a prognostic marker for overall survival and response to therapy in colon cancer.

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    Cannan, W. J.; Pederson, D. S. Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin. J. Cell. Physiol. 2016, 231, 3– 14,  DOI: 10.1002/jcp.25048

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    Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin

    Cannan, Wendy J.; Pederson, David S.

    Journal of Cellular Physiology (2016), 231 (1), 3-14CODEN: JCLLAX; ISSN:0021-9541. (Wiley-Blackwell)

    A review. All organisms suffer double-strand breaks (DSBs) in their DNA as a result of exposure to ionizing radiation. DSBs can also form when replication forks encounter DNA lesions or repair intermediates. The processing and repair of DSBs can lead to mutations, loss of heterozygosity, and chromosome rearrangements that result in cell death or cancer. The most common pathway used to repair DSBs in metazoans (non-homologous DNA end joining) is more commonly mutagenic than the alternative pathway (homologous recombination mediated repair). Thus, factors that influence the choice of pathways used DSB repair can affect an individual's mutation burden and risk of cancer. This review describes radiol., chem., and biol. mechanisms that generate DSBs, and discusses the impact of such variables as DSB etiol., cell type, cell cycle, and chromatin structure on the yield, distribution, and processing of DSBs. The final section focuses on nucleosome-specific mechanisms that influence DSB prodn., and the possible relationship between higher order chromosome coiling and chromosome shattering (chromothripsis).

27. 27

    Eccles, L. J.; O’Neill, P.; Lomax, M. E. Delayed Repair of Radiation Induced Clustered DNA Damage: Friend or Foe?. Mutat. Res. - Fundam. Mol. Mec. 2011, 711, 134– 141,  DOI: 10.1016/j.mrfmmm.2010.11.003

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    Delayed repair of radiation induced clustered DNA damage: Friend or foe?

    Eccles, Laura J.; O'Neill, Peter; Lomax, Martine E.

    Mutation Research, Fundamental and Molecular Mechanisms of Mutagenesis (2011), 711 (1-2), 134-141CODEN: MUREAV; ISSN:0027-5107. (Elsevier B.V.)

    A review. A signature of ionizing radiation exposure is the induction of DNA clustered damaged sites, defined as two or more lesions within one to two helical turns of DNA by passage of a single radiation track. Clustered damage is made up of double strand breaks (DSB) with assocd. base lesions or abasic (AP) sites, and non-DSB clusters comprised of base lesions, AP sites and single strand breaks. This review will conc. on the exptl. findings of the processing of non-DSB clustered damaged sites. It has been shown that non-DSB clustered damaged sites compromise the base excision repair pathway leading to the lifetime extension of the lesions within the cluster, compared to isolated lesions, thus the likelihood that the lesions persist to replication and induce mutation is increased. In addn. certain non-DSB clustered damaged sites are processed within the cell to form addnl. DSB. The use of E. coli to demonstrate that clustering of DNA lesions is the major cause of the detrimental consequences of ionizing radiation is also discussed. The delayed repair of non-DSB clustered damaged sites in humans can be seen as a "friend", leading to cell killing in tumor cells or as a "foe", resulting in the formation of mutations and genetic instability in normal tissue.

    Thompson, P. S.; Cortez, D. New Insights into Abasic Site Repair and Tolerance. DNA repair 2020, 90, 102866,  DOI: 10.1016/j.dnarep.2020.102866

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    New insights into abasic site repair and tolerance

    Thompson, Petria S.; Cortez, David

    DNA Repair (2020), 90 (), 102866CODEN: DRNEAR; ISSN:1568-7864. (Elsevier B.V.)

    A review. Thousands of apurinic/apyrimidinic (AP or abasic) sites form in each cell, each day. This simple DNA lesion can have profound consequences to cellular function, genome stability, and disease. As potent blocks to polymerases, they interfere with the reading and copying of the genome. Since they provide no coding information, they are potent sources of mutation. Due to their reactive chem., they are intermediates in the formation of lesions that are more challenging to repair including double-strand breaks, interstrand crosslinks, and DNA protein crosslinks. Given their prevalence and deleterious consequences, cells have multiple mechanisms of repairing and tolerating these lesions. While base excision repair of abasic sites in double-strand DNA has been studied for decades, new interest in abasic site processing has come from more recent insights into how they are processed in single-strand DNA. In this review, we discuss the source of abasic sites, their biol. consequences, tolerance mechanisms, and how they are repaired in double and single-stranded DNA.

28. 28

    Mah, L.; El-Osta, A.; Karagiannis, T. γH2AX: A Sensitive Molecular Marker of DNA Damage and Repair. Leukemia 2010, 24, 679– 686,  DOI: 10.1038/leu.2010.6

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    γH2AX: a sensitive molecular marker of DNA damage and repair

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    Leukemia (2010), 24 (4), 679-686CODEN: LEUKED; ISSN:0887-6924. (Nature Publishing Group)

    A review. Phosphorylation of the Ser-139 residue of the histone variant H2AX, forming γH2AX, is an early cellular response to the induction of DNA double-strand breaks. Detection of this phosphorylation event has emerged as a highly specific and sensitive mol. marker for monitoring DNA damage initiation and resoln. Further, anal. of γH2AX foci has numerous other applications including, but not limited to, cancer and aging research. Quantitation of γH2AX foci has also been applied as a useful tool for the evaluation of the efficacy of various developmental drugs, particularly, radiation modifying compds. This review focuses on the current status of γH2AX as a marker of DNA damage and repair in the context of ionizing radiation. Although the emphasis is on γ-radiation-induced γH2AX foci, the effects of other genotoxic insults including exposure to UV rays, oxidative stress and chem. agents are also discussed.

29. 29

    Haskins, J. S.; Su, C.; Maeda, J.; Walsh, K. D.; Haskins, A. H.; Allum, A. J.; Froning, C. E.; Kato, T. A. Evaluating the Genotoxic and Cytotoxic Effects of Thymidine Analogs, 5-Ethynyl-2′-Deoxyuridine and 5-Bromo-2′-Deoxyurdine to Mammalian Cells. Int. J. Mol. Sci. 2020, 21, 6631,  DOI: 10.3390/ijms21186631

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    Evaluating the genotoxic and cytotoxic effects of thymidine analogs, 5-ethynyl-2-deoxyuridine and 5-bromo-2-deoxyurdine to mammalian cells

    Haskins, Jeremy S.; Su, Cathy; Maeda, Junko; Walsh, Kade D.; Haskins, Alexis H.; Allum, Allison J.; Froning, Coral E.; Kato, Takamitsu A.

    International Journal of Molecular Sciences (2020), 21 (18), 6631CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)

    BrdU (bromodeoxyuridine) and EdU (ethynyldeoxyuridine) have been largely utilized as the means of monitoring DNA replication and cellular division. Although BrdU induces gene and chromosomal mutations and induces sensitization to photons, EdU's effects have not been extensively studied yet. Therefore, we investigated EdU's potential cytotoxic and mutagenic effects and its related underlying mechanisms when administered to Chinese hamster ovary (CHO) wild type and DNA repair-deficient cells. EdU treatment displayed a higher cytotoxicity and genotoxicity than BrdU treatment. Cells with defective homologous recombination repair displayed a greater growth delay and severe inhibition of clonogenicity with EdU compared to wild type and other DNA repair-deficient cells. Inductions of sister chromatid exchange and hypoxanthine phosphorybosyl transferase (HPRT) mutation were obsd. in EdU-incorporated cells as well. Interestingly, on the other hand, EdU did not induce sensitization to photons to the same degree as BrdU. Our results demonstrate that elevated concns. (similar to manufacturers suggested concn.; >5-10μM) of EdU treatment were toxic to the cell cultures, particularly in cells with a defect in homologous recombination repair. Therefore, EdU should be administered with addnl. precautions.

30. 30

    LeBlanc, D. P.; Meier, M.; Lo, F. Y.; Schmidt, E.; Valentine, C.; Williams, A.; Salk, J. J.; Yauk, C. L.; Marchetti, F. Duplex Sequencing Identifies Genomic Features that Determine Susceptibility to Benzo[a]pyrene-Induced in vivo Mutations. BMC Genomics 2022, 23, 542,  DOI: 10.1186/s12864-022-08752-w

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    Duplex sequencing identifies genomic features that determine susceptibility to benzo(a)pyrene-induced in vivo mutations

    LeBlanc, Danielle P. M.; Meier, Matthew; Lo, Fang Yin; Schmidt, Elizabeth; Valentine III, Charles; Williams, Andrew; Salk, Jesse J.; Yauk, Carole L.; Marchetti, Francesco

    BMC Genomics (2022), 23 (1), 542CODEN: BGMEET; ISSN:1471-2164. (BioMed Central Ltd.)

    Exposure to environmental mutagens increases the risk of cancer and genetic disorders. We used Duplex Sequencing (DS), a high-accuracy error-cor. sequencing technol., to analyze mutation induction across twenty 2.4 kb intergenic and genic targets in the bone marrow of MutaMouse males exposed to benzo(a)pyrene (BaP), a widespread environmental pollutant. DS revealed a linear dose-related induction of mutations across all targets with low intra-group variability. Heterochromatic and intergenic regions exhibited the highest mutation frequencies (MF). C:G > A:T transversions at CCA, CCC and GCC trinucleotides were enriched in BaP-exposed mice consistent with the known etiol. of BaP mutagenesis. However, GC-content had no effect on mutation susceptibility. A pos. correlation was obsd. between DS and the "gold-std." transgenic rodent gene mutation assay. Overall, we demonstrate that DS is a promising approach to study in vivo mutagenesis and yields crit. insight into the genomic features governing mutation susceptibility, spectrum, and variability across the genome.

31. 31

    Theisinger, A.; Grenacher, B.; Rech, K.; Scharrer, E. Nucleosides are Efficiently Absorbed by Na-Dependent Transport Across the Intestinal Brush Border Membrane in Veal Calves. J. Dairy Sci. 2002, 85, 2308– 2314,  DOI: 10.3168/jds.S0022-0302(02)74311-7

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    Nucleosides are efficiently absorbed by Na+-dependent transport across the intestinal brush border membrane in veal calves

    Theisinger, Anja; Grenacher, B.; Rech, K. S.; Scharrer, E.

    Journal of Dairy Science (2002), 85 (9), 2308-2314CODEN: JDSCAE; ISSN:0022-0302. (American Dairy Science Association)

    In previous work, a comparatively high capacity for Na+-dependent transport of nucleosides across the intestinal brush border membrane (BBM) was obsd. in dairy cows, which might be related to digestion of the large amt. of nucleic acids present in ruminal microorganisms in the ruminant small intestine. If this were the case, the capacity for Na+-dependent intestinal nucleoside transport should be much lower in veal calves, in which only small amts. of nucleic acids, nucleotides, and nucleosides reach the small intestine via the milk replacer. To test this hypothesis, we investigated Na+-dependent transport of 3H-labeled thymidine and guanosine across the BBM using BBM vesicles (BBMV) isolated from the small intestine of veal calves. In the presence of a transmembrane Na+ gradient both substrates were transported against a concn. gradient. Inhibitory studies showed that thymidine and guanosine are transported by two different transporters with overlapping substrate specificity, one accepting predominantly pyrimidine nucleosides (N2) and one accepting particularly purine nucleosides (N1). Nucleoside transport was inhibited by glucose along the whole small intestine. Maximal transport rates similar to those in dairy cows were obtained for the proximal, mid-, and distal small intestine. These findings suggest that the high absorptive capacity for nucleosides is a genetically fixed property in the bovine small intestine, which is already present in the preruminant state of veal calves. It may contribute to the high digestibility of nucleic acids obsd. by others in veal calves receiving milk replacer supplemented with RNA. Its main function may be the efficient absorption of nucleosides resulting from the digestion of nucleic acids assocd. with desquamated enterocytes. Due to the limited de novo synthesis of nucleotides in enterocytes intracellular uptake of nucleosides across the BBM may contribute to nucleic acid synthesis in enterocytes and thus may have a trophic effect on the intestinal epithelium.

32. 32

    Cadet, J.; Wagner, J. R. DNA Base Damage by Reactive Oxygen Species, Oxidizing Agents, and UV Radiation. Cold Spring Harb. Perspect. Biol. 2013, 5, a012559,  DOI: 10.1101/cshperspect.a012559

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    DNA base damage by reactive oxygen species, oxidizing agents, and UV radiation

    Cadet, Jean; Wagner, J. Richard

    Cold Spring Harbor Perspectives in Biology (2013), 5 (2), A012559/1-A012559/18CODEN: CSHPEU; ISSN:1943-0264. (Cold Spring Harbor Laboratory Press)

    A review. Emphasis has been placed in this article dedicated to DNA damage on recent aspects of the formation and measurement of oxidatively generated damage in cellular DNA in order to provide a comprehensive and updated survey. This includes single pyrimidine and purine base lesions, intrastrand crosslinks, purine 5',8-cyclonucleosides, DNA-protein adducts and interstrand crosslinks formed by the reactions of either the nucleobases or the 2-deoxyribose moiety with the hydroxyl radical, one-electron oxidants, singlet oxygen, and hypochlorous acid. In addn., recent information concerning the mechanisms of formation, individual measurement, and repair-rate assessment of bipyrimidine photoproducts in isolated cells and human skin upon exposure to UVB radiation, UVA photons, or solar simulated light is critically reviewed.

33. 33

    Chen, Y.; Liu, X.; Sun, X.; Zhang, J.; Mi, Y.; Li, Q.; Guo, Z. Synthesis and Antioxidant Activity of Cationic 1, 2, 3-Triazole Functionalized Starch Derivatives. Polymers 2020, 12, 112,  DOI: 10.3390/polym12010112

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    Synthesis and antioxidant activity of cationic 1,2,3-triazole functionalized starch derivatives

    Chen, Yuan; Liu, Xiguang; Sun, Xueqi; Zhang, Jingjing; Mi, Yingqi; Li, Qing; Guo, Zhanyong

    Polymers (Basel, Switzerland) (2020), 12 (1), 112CODEN: POLYCK; ISSN:2073-4360. (MDPI AG)

    In this study, starch was chem. modified to improve its antioxidant activity. Five novel cationic 1,2,3-triazole functionalized starch derivs. were synthesized by using "click" reaction and N-alkylation. A convenient method for pre-azidation of starch was developed. The structures of the derivs. were analyzed using FTIR and 1H NMR. The radicals scavenging abilities of the derivs. against hydroxyl radicals, DPPH radicals, and superoxide radicals were tested in vitro in order to evaluate their antioxidant activity. Results revealed that all the cationic starch derivs. (2a-2e), as well as the precursor starch derivs. (1a-1e), had significantly improved antioxidant activity compared to native starch. In particular, the scavenging ability of the derivs. against superoxide radicals was extremely strong. The improved antioxidant activity benefited from the enhanced soly. and the added pos. charges. The biocompatibility of the cationic derivs. was confirmed by the low hemolytic rate (<2%). The obtained derivs. in this study have great potential as antioxidant materials that can be applied in the fields of food and biomedicine.

    Yang, S.; Guo, Z.; Miao, F.; Xue, Q.; Qin, S. The Hydroxyl Radical Scavenging Activity of Chitosan, Hyaluronan, Starch and Their O-Carboxymethylated Derivatives. Carbohydr. Polym. 2010, 82, 1043– 1045,  DOI: 10.1016/j.carbpol.2010.06.014

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    The hydroxyl radical scavenging activity of chitosan, hyaluronan, starch and their O-carboxymethylated derivatives

    Yang, Shaoli; Guo, Zhanyong; Miao, Fengping; Xue, Qinzhao; Qin, Song

    Carbohydrate Polymers (2010), 82 (4), 1043-1045CODEN: CAPOD8; ISSN:0144-8617. (Elsevier Ltd.)

    In order to study the effect of active hydroxyl and amino groups on scavenging activity against hydroxyl radicals of polysaccharides, three kinds of carboxymethylated polysaccharides (carboxymethyl chitosan (O-CM-chitosan), carboxymethyl hyaluronan (CMHA), and carboxymethyl starch (CMS)) were prepd. and their antioxidant activities against hydroxyl radicals were assessed, resp. Results showed that O-CM-chitosan had lower scavenging ability on hydroxyl radicals than chitosan. CMHA and CMS had the same tendency. For the three kinds of polysaccharides, scavenging ability on hydroxyl radicals was found to be in the order of chitosan > HA > starch. The scavenging ability of carboxymethylated polysaccharides had the same order as related to its corresponding polysaccharides at higher concns. (≥0.8 mg/mL). There were not only hydroxyl groups but also amino or acetamino (CH3CONH-) groups in the mols. of chitosan and HA, but only hydroxyl group for starch. It was suggested that the sequence influence the scavenging activity against hydroxyl radicals might be amino group > acetamide group > hydroxyl group.

34. 34

    Watling, C. Z.; Schmidt, J. A.; Dunneram, Y.; Tong, T. Y.; Kelly, R. K.; Knuppel, A.; Travis, R. C.; Key, T. J.; Perez-Cornago, A. Risk of Cancer in Regular and Low Meat-Eaters, Fish-Eaters, and Vegetarians: a Prospective Analysis of UK Biobank Participants. BMC Medicine 2022, 20, 73,  DOI: 10.1186/s12916-022-02256-w

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    Risk of cancer in regular and low meat-eaters, fish-eaters, and vegetarians: a prospective analysis of UK Biobank participants

    Watling Cody Z; Schmidt Julie A; Dunneram Yashvee; Tong Tammy Y N; Kelly Rebecca K; Knuppel Anika; Travis Ruth C; Key Timothy J; Perez-Cornago Aurora

    BMC medicine (2022), 20 (1), 73 ISSN:.

    BACKGROUND: Following a vegetarian diet has become increasingly popular and some evidence suggests that being vegetarian may be associated with a lower risk of cancer overall. However, for specific cancer sites, the evidence is limited. Our aim was to assess the associations of vegetarian and non-vegetarian diets with risks of all cancer, colorectal cancer, postmenopausal breast cancer, and prostate cancer and to explore the role of potential mediators between these associations. METHODS: We conducted a prospective analysis of 472,377 UK Biobank participants who were free from cancer at recruitment. Participants were categorised into regular meat-eaters (n = 247,571), low meat-eaters (n = 205,385), fish-eaters (n = 10,696), and vegetarians (n = 8685) based on dietary questions completed at recruitment. Multivariable-adjusted Cox regressions were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for all cancer incidence and separate cancer sites across diet groups. RESULTS: After an average follow-up of 11.4 years, 54,961 incident cancers were identified, including 5882 colorectal, 7537 postmenopausal breast, and 9501 prostate cancers. Compared with regular meat-eaters, being a low meat-eater, fish-eater, or vegetarian were all associated with a lower risk of all cancer (HR: 0.98, 95% CI: 0.96-1.00; 0.90, 0.84-0.96; 0.86, 0.80-0.93, respectively). Being a low meat-eater was associated with a lower risk of colorectal cancer in comparison to regular meat-eaters (0.91, 0.86-0.96); however, there was heterogeneity in this association by sex (p = 0.007), with an inverse association across diet groups in men, but not in women. Vegetarian postmenopausal women had a lower risk of breast cancer (0.82, 0.68-0.99), which was attenuated and non-significant after adjusting for body mass index (BMI; 0.87, 0.72-1.05); in mediation analyses, BMI was found to possibly mediate the observed association. In men, being a fish-eater or a vegetarian was associated with a lower risk of prostate cancer (0.80, 0.65-0.99 and 0.69, 0.54-0.89, respectively). CONCLUSION: The lower risk of colorectal cancer in low meat-eaters is consistent with previous evidence suggesting an adverse impact of meat intake. The lower risk of postmenopausal breast cancer in vegetarian women may be explained by their lower BMI. It is not clear whether the other differences observed for all cancers and for prostate cancer reflect any causal relationships or are due to other factors such as residual confounding or differences in cancer detection.

35. 35

    Nagy, G. N.; Leveles, I.; Vértessy, B. G. Preventive DNA Repair by Sanitizing the Cellular (Deoxy) Nucleoside Triphosphate Pool. FEBS journal 2014, 281, 4207– 4223,  DOI: 10.1111/febs.12941

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    35

    Preventive DNA repair by sanitizing the cellular (deoxy)nucleoside triphosphate pool

    Nagy, Gergely N.; Leveles, Ibolya; Vertessy, Beata G.

    FEBS Journal (2014), 281 (18), 4207-4223CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)

    A review. The occurrence of modified bases in DNA is attributed to some major factors: incorporation of altered nucleotide building blocks and chem. reactions or radiation effects on bases within the DNA structure. Several 'house-cleaning' enzyme families are involved in preventing the incorporation of noncanonical bases playing a 'sanitizing' role. The catalytic mechanism of action of these enzymes has been revealed for a no. of representatives in clear structural and kinetic detail. Here, the authors focus in detail on those examples where clear evidence has been produced using high-resoln. structural studies. Comparing the protein fold and architecture of the enzyme active sites, 2 main classes of sanitizing deoxyribonucleoside triphosphate pyrophosphatases can be assigned that are distinguished by the site of nucleophilic attack. In enzymes assocd. with attack at the α-P atom, it is shown that coordination of the γ-phosphate group is also ensured by multiple interactions. By contrast, enzymes catalyzing attack at the β-P atom mainly coordinate the α- and the β-phosphate only. Characteristic differences are also obsd. with respect to the role of the metal ion cofactor (Mg2+) and the coordination of nucleophilic water. Using different catalytic mechanisms embedded in different protein folds, these enzymes present a clear example of convergent evolution.

36. 36

    Kiwerska, K.; Szyfter, K. DNA Repair in Cancer Initiation, Progression, and Therapy─a Double-Edged Sword. J. Appl. Genet. 2019, 60, 329– 334,  DOI: 10.1007/s13353-019-00516-9

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    DNA repair in cancer initiation, progression, and therapy-a double-edged sword

    Kiwerska, Katarzyna; Szyfter, Krzysztof

    Journal of Applied Genetics (2019), 60 (3-4), 329-334CODEN: JAGEFC; ISSN:1234-1983. (Springer GmbH)

    Genomic and mitochondrial DNA mols. are exposed continuously for a damaging activity of chem., phys., and internal genotoxicants. When DNA repair machinery is not working efficiently, the generation of DNA lesions and mutations leads to carcinogenic transformation. The high no. of mutation going up to 105 per cell was recognized as a driving force of oncogenesis. Moreover, a high activity of DNA repair genes was hypothesized as a predisposition to metastasis. DNA repair potential has to be taken into account attempting to chemo- and/or radiotherapy. A low activity of DNA repair genes makes tumor cells more sensitive to therapy, but on the other hand, non-tumor cells getting lesions could form second primary cancer. Contrary, high activity of DNA repair genes counteracts attempted therapy. It means an individualized therapy based on recognition of DNA repair potential is recommended.

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• This article references 36 other publications.

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     Meat cooking methods and risk of type 2 diabetes: results from three prospective cohort studies

     Liu, Gang; Zong, Geng; Wu, Kana; Hu, Yang; Li, Yanping; Willett, Walter C.; Eisenberg, DavidM.; Hu, Frank B.; Sun, Qi

     Diabetes Care (2018), 41 (5), 1049-1060CODEN: DICAD2; ISSN:0149-5992. (American Diabetes Association, Inc.)

     OBJECTIVE To examine open-flame and/or high-temp. cooking (grilling/barbecuing, broiling, or roasting) and doneness preferences (rare ,medium, or well done) for redmeat, chicken, and fish in relation to type 2 diabetes (T2D) risk among U.S. adults who consumed animal flesh regularly (≥2 servings/wk). RESEARCH DESIGN AND METHODS The prospective studies included 52,752women from the Nurses' Health Study (NHS) (followed during 1996-2012), 60,809 women from NHS II (followed during 2001- 2013), and 24,679 men from the Health Professionals Follow-Up Study (HPFS) (followed during 1996-2012) who were free of diabetes, cardiovascular disease, and cancer at baseline. Incident cases of T2D were confirmed by validated supplementary questionnaires. RESULTS We documented 7,895 incident cases of T2D during 1.74 million person-years of follow-up. Aftermultivariate adjustments including baseline BMI and total consumption of red meat, chicken, and fish, higher frequency of open-flame and/or high temp. cooking was independently assocd. with an elevated T2D risk. When comparing open-flame and/or high-temp. cooking >15 times/mo with <4 times/mo, the pooled hazard ratio (HR) (95% CI) of T2D was 1.28 (1.18, 1.39; Ptrend <0.001). When comparing the extreme quartiles of doneness-weighted frequency of high-temp. cooking, the pooled HR (95% CI) of T2D was 1.20 (1.12, 1.28; Ptrend <0.001). These assocns. remained significant when red meat and chicken were examd. sep. In addn., estd. intake of heterocyclic arom. amines was also assocd. with an increased T2D risk. CONCLUSIONS Independent of consumption amt., open-flame and/or high-temp. cooking for both redmeat and chicken is assocd. with an increased T2D risk among adults who consume animal flesh regularly.

  3. 3

     Ramirez-Anaya, J. d. P.; Samaniego-Sanchez, C.; Castaneda-Saucedo, M. C.; Villalon-Mir, M.; de la Serrana, H. L.-G. Phenols and the Antioxidant Capacity of Mediterranean Vegetables Prepared with Extra Virgin Olive Oil Using Different Domestic Cooking Techniques. Food chemistry 2015, 188, 430– 438,  DOI: 10.1016/j.foodchem.2015.04.124

     3

     Phenols and the antioxidant capacity of Mediterranean vegetables prepared with extra virgin olive oil using different domestic cooking techniques

     Ramirez-Anaya, Jessica del Pilar; Samaniego-Sanchez, Cristina; Castaneda-Saucedo, Ma. Claudia; Villalon-Mir, Marina; de la Serrana, Herminia Lopez-Garcia

     Food Chemistry (2015), 188 (), 430-438CODEN: FOCHDJ; ISSN:0308-8146. (Elsevier Ltd.)

     Potato, tomato, eggplant and pumpkin were deep fried, sauteed and boiled in Mediterranean extra virgin olive oil (EVOO), water, and a water/oil mixt. (W/O). The authors detd. the contents of fat, moisture, total phenols (TPC) and eighteen phenolic compds., as well as antioxidant capacity in the raw vegetables and compared these with contents measured after cooking. Deep frying and sauteing led to increased fat contents and TPC, whereas both types of boiling (in water and W/O) reduced the same. The presence of EVOO in cooking increased the phenolics identified in the raw foods as oleuropein, pinoresinol, hydroxytyrosol and tyrosol, and the contents of vegetable phenolics such as chlorogenic acid and rutin. All the cooking methods conserved or increased the antioxidant capacity measured by DPPH, FRAP and ABTS. Multivariate analyses showed that each cooked vegetable developed specific phenolic and antioxidant activity profiles resulting from the characteristics of the raw vegetables and the cooking techniques.

  4. 4

     Shaughnessy, D. T.; Gangarosa, L. M.; Schliebe, B.; Umbach, D. M.; Xu, Z.; MacIntosh, B.; Knize, M. G.; Matthews, P. P.; Swank, A. E.; Sandler, R. S. Inhibition of Fried Meat-Induced Colorectal DNA Damage and Altered Systemic Genotoxicity in Humans by Crucifera, Chlorophyllin, and Yogurt. PloS one 2011, 6, e18707,  DOI: 10.1371/journal.pone.0018707

     4

     Inhibition of fried meat-induced colorectal DNA damage and altered systemic genotoxicity in humans by crucifera, chlorophyllin, and yogurt

     Shaughnessy, Daniel T.; Gangarosa, Lisa M.; Schliebe, Barbara; Umbach, David M.; Xu, Zongli; MacIntosh, Beth; Knize, Mark G.; Matthews, Peggy P.; Swank, Adam E.; Sandler, Robert S.; DeMarini, David M.; Taylor, Jack A.

     PLoS One (2011), 6 (4), e18707CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)

     Dietary exposures implicated as reducing or causing risk for colorectal cancer may reduce or cause DNA damage in colon tissue; however, no one has assessed this hypothesis directly in humans. Thus, we enrolled 16 healthy volunteers in a 4-wk controlled feeding study where 8 subjects were randomly assigned to dietary regimens contg. meat cooked at either low (100°C) or high temp. (250°C), each for 2 wk in a crossover design. The other 8 subjects were randomly assigned to dietary regimens contg. the high-temp. meat diet alone or in combination with 3 putative mutagen inhibitors: cruciferous vegetables, yogurt, and chlorophyllin tablets, also in a crossover design. Subjects were nonsmokers, at least 18 years old, and not currently taking prescription drugs or antibiotics. We used the Salmonella assay to analyze the meat, urine, and feces for mutagenicity, and the comet assay to analyze rectal biopsies and peripheral blood lymphocytes for DNA damage. Low-temp. meat had undetectable levels of heterocyclic amines (HCAs) and was not mutagenic, whereas high-temp. meat had high HCA levels and was highly mutagenic. The high-temp. meat diet increased the mutagenicity of hydrolyzed urine and feces compared to the low-temp. meat diet. The mutagenicity of hydrolyzed urine was increased nearly twofold by the inhibitor diet, indicating that the inhibitors enhanced conjugation. Inhibitors decreased significantly the mutagenicity of un-hydrolyzed and hydrolyzed feces. The diets did not alter the levels of DNA damage in non-target white blood cells, but the inhibitor diet decreased nearly twofold the DNA damage in target colorectal cells. To our knowledge, this is the first demonstration that dietary factors can reduce DNA damage in the target tissue of fried-meat assocd. carcinogenesis.

  5. 5

     Colvin, M. E.; Hatch, F. T.; Felton, J. S. Chemical and Biological Factors Affecting Mutagen Potency. Mutat. Res. 1998, 400, 479– 492,  DOI: 10.1016/S0027-5107(98)00073-6

     5

     Chemical and biological factors affecting mutagen potency

     Colvin, Michael E.; Hatch, Frederick T.; Felton, James S.

     Mutation Research, Fundamental and Molecular Mechanisms of Mutagenesis (1998), 400 (1,2), 479-492CODEN: MUREAV; ISSN:0027-5107. (Elsevier Science B.V.)

     A review with 72 refs. on the chem. and biol. factors that are correlated with the mutagenic activity of the arom. and heterocyclic amines. Particular attention is given to the predicted quantum chem. properties of the parent amines and their metabolites. A no. of chem. properties have been found to correlate well with measured mutagenic potency, including log P, the energies of the frontier orbitals of the parent amines, and the thermodn. stability of the nitrenium ion, possibly the ultimate DNA-binding species. These correlations are intriguing clues to the mutagenic activity of the arom. amines; however, many factors still await final explanation, including the exact mechanisms of the metabolic enzymes, the identity(s) of the ultimate DNA-binding species, the reaction mechanism in the DNA-adduction, the role of sequence context in the covalent and non-covalent binding of the adducts, and the role of DNA repair.

  6. 6

     Wu, J.; Xiao, J.; Zhang, Z.; Wang, X.; Hu, S.; Yu, J. Ribogenomics: the Science and Knowledge of RNA. Genomics, proteomics & bioinformatics 2014, 12, 57– 63,  DOI: 10.1016/j.gpb.2014.04.002

     6

     Ribogenomics: the science and knowledge of RNA

     Wu, Jiayan; Xiao, Jingfa; Zhang, Zhang; Wang, Xumin; Hu, Songnian; Yu, Jun

     Genomics, Proteomics & Bioinformatics (2014), 12 (2), 57-63CODEN: GPBEBL; ISSN:1672-0229. (Science Press)

     A review. RNA (RNA) deserves not only a dedicated field of biol. research-a discipline or branch of knowledge-but also explicit definitions of its roles in cellular processes and mol. mechanisms. Ribogenomics is to study the biol. of cellular RNAs, including their origin, biogenesis, structure and function. On the informational track, mRNAs (mRNAs) are the major component of ribogenomes, which encode proteins and serve as one of the four major components of the translation machinery and whose expression is regulated at multiple levels by other operational RNAs. On the operational track, there are several diverse types of RNAs-their length distribution is perhaps the most simplistic stratification-involving in major cellular activities, such as chromosomal structure and organization, DNA replication and repair, transcriptional/post-transcriptional regulation, RNA processing and routing, translation and cellular energy/metab. regulation. An all-out effort exceeding the magnitude of the Human Genome Project is of essence to construct just mammalian transcriptomes in multiple contexts including embryonic development, circadian and seasonal rhythms, defined life-span stages, pathol. conditions and anatomy-driven tissue/organ/cell types.

  7. 7

     Lindahl, T.; Nyberg, B. Heat-Induced Deamination of Cytosine Residues in Deoxyribonucleic Acid. Biochemistry 1974, 13, 3405– 3410,  DOI: 10.1021/bi00713a035

     7

     Heat-induced deamination of cytosine residues in deoxyribonucleic acid

     Lindahl, Tomas; Nyberg, Barbro

     Biochemistry (1974), 13 (16), 3405-10CODEN: BICHAW; ISSN:0006-2960.

     The rate of deamination of cytosine residues in single-stranded and double-stranded Escherichia coli DNA, in the polynucleotides poly(dC) and poly(dG)-poly(dC), and in dCMP was investigated as a function of temp., pH, and buffer compn. For this purpose, nucleic acids and polydeoxynucleotides specifically radioactively labeled in the cytosine residues were prepd. After heat treatment, the polymers were enzymically degraded to mononucleotides or nucleosides, cytosine and uracil derivs. were sepd. by paper chromatog., and their radioactivity was detd. Cytosine in single-stranded DNA, poly(dC), or dCMP is similarly susceptible to hydrolytic deamination at pH 7.4 and the reaction proceeds at a rate of k = 2 × 10-7 sec-1 at 95°. From measurements at several temps. it is estd. that the reaction is assocd. with an activation energy of 29 kcal/mole. These data indicate that a significant amt. of conversion of cytosine to uracil occurs during heat denaturation of DNA by std. procedures. The cytosine residues in native DNA are well protected and are deaminated at <1% of the rate obsd. with dCMP or poly(dC). In contrast, the cytosine residues in poly(dG)-poly(dC) were deaminated at 75% of the rate of those in poly(dC). The in vivo rate of deamination of cytosine residues in DNA is discussed.

  8. 8

     Bruskov, V. I.; Malakhova, L. V.; Masalimov, Z. K.; Chernikov, A. V. Heat-Induced Formation of Reactive Oxygen Species and 8-Oxoguanine, a Biomarker of Damage to DNA. Nucleic Acids Res. 2002, 30, 1354– 1363,  DOI: 10.1093/nar/30.6.1354

     8

     Heat-induced formation of reactive oxygen species and 8-oxoguanine, a biomarker of damage to DNA

     Bruskov, Vadim I.; Malakhova, Lyudmila V.; Masalimov, Zhaksylyk K.; Chernikov, Anatoly V.

     Nucleic Acids Research (2002), 30 (6), 1354-1363CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)

     Heat-induced formation of 8-oxoguanine was demonstrated in DNA solns. in 10-3 M phosphate buffer, pH 6.8, by enzyme-linked immunosorbent assays using monoclonal antibodies against 8-oxoguanine. A radiation-chem. yield of 3.7 × 10-2 μmol J-1 for 8-oxoguanine prodn. in DNA upon γ-irradn. was used as an adequate std. for quantitation of 8-oxoguanine in whole DNA. The initial yield of heat-induced 8-oxoguanine exhibits first order kinetics. The rate consts. for 8-oxoguanine formation were detd. at elevated temps.; the activation energy was found to be 27 ± 2 kcal/mol. Extrapolation to 37°C gave a value of k37 = 4.7 × 10-10 s-1. Heat-induced 8-oxoguanine formation and depurination of guanine and adenine show similarities of the processes, which implies that heat-mediated generation of reactive oxygen species (ROS) should occur. Heat-induced prodn. of H2O2 in phosphate buffer was shown. The sequence of reactions of thermally mediated ROS formation have been established: activation of dissolved oxygen to the singlet state, generation of superoxide radicals and their dismutation to H2O2. Gas satn. (O2, N2 and Ar), D2O, scavengers of 1O2, O2- and OH. radicals and metal chelators influenced heat-induced 8-oxoguanine formation as they affected thermal ROS generation. These findings imply that heat acts via ROS attack leading to oxidative damage to DNA.

  9. 9

     Jonas, D.; Elmadfa, I.; Engel, K.-H.; Heller, K.; Kozianowski, G.; König, A.; Müller, D.; Narbonne, J.; Wackernagel, W.; Kleiner, J. Safety Considerations of DNA in Food. Ann. Nutr. Metab. 2001, 45, 235– 254,  DOI: 10.1159/000046734

     9

     Safety Considerations of DNA in Food

     Jonas, D. A.; Elmadfa, I.; Engel, K.-H.; Heller, K. J.; Kozianowski, G.; Koenig, A.; Mueller, D.; Narbonne, J. F.; Wackernagel, W.; Kleiner, J.

     Annals of Nutrition & Metabolism (2001), 45 (6), 235-254CODEN: ANUMDS; ISSN:0250-6807. (S. Karger AG)

     A review. Recombinant DNA techniques are capable of introducing genetic changes into food organisms that are more predictable than those introduced through conventional breeding techniques. This review discusses whether the consumption of DNA in approved novel foods and novel food ingredients derived from genetically modified organisms (GMOs) can be regarded as being as safe as the consumption of DNA in existing foods. It concludes that DNA from GMOs is equiv. to DNA from existing food organisms that has always been consumed with human diets. Any risks assocd. with the consumption of DNA will remain, irresp. of its origin, because the body handles all DNA in the same way. The breakdown of DNA during food processing and passage through the gastrointestinal tract reduces the likelihood that intact genes capable of encoding foreign proteins will be transferred to gut microflora. The review does not specifically address food safety issues arising from the consumption of viable genetically modified microorganisms but it shows that the likelihood of transfer and functional integration of DNA from ingested food by gut microflora and/or human cells is minimal. Information reviewed does not indicate any safety concerns assocd. with the ingestion of DNA per se from GMOs resulting from the use of currently available recombinant DNA techniques in the food chain.

  10. 10

      Carver, J. D.; Walker, W. A. The Role of Nucleotides in Human Nutrition. J. Nutr. Biochem 1995, 6, 58– 72,  DOI: 10.1016/0955-2863(94)00019-I

      10

      The role of nucleotides in human nutrition

      Carver, Jane D.; Walker, W. Allan

      Journal of Nutritional Biochemistry (1995), 6 (2), 58-72CODEN: JNBIEL; ISSN:0955-2863. (Elsevier)

      A review with 182 refs. on nucleotide occurrence, biochem., absorption and metab., and internal effects, and the use of nucleotides as nutritional supplements.

  11. 11

      Henderson, P. T.; Evans, M. D.; Cooke, M. S. Salvage of Oxidized Guanine Derivatives in the (2′-deoxy) Ribonucleotide Pool as Source of Mutations in DNA. Mutat. Res. Genet. Toxicol. Environ. Mutagen. 2010, 703, 11– 17,  DOI: 10.1016/j.mrgentox.2010.08.021

      11

      Salvage of oxidized guanine derivatives in the (2'-deoxy)ribonucleotide pool as source of mutations in DNA

      Henderson, Paul T.; Evans, Mark D.; Cooke, Marcus S.

      Mutation Research, Genetic Toxicology and Environmental Mutagenesis (2010), 703 (1), 11-17CODEN: MRGMFI; ISSN:1383-5718. (Elsevier B.V.)

      A review. Recent evidence suggests that salvage of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydro-guanine (8-oxoGua) can contribute substantially to levels of 8-oxoGua in DNA and RNA. However, it remains to be detd. if this mechanism contributes to mutagenesis and disease. This review covers the predominant methods for detecting 8-oxoGua and its derivs., summarizes some of the relevant recent DNA repair studies and discusses the mechanisms for metab. of oxidized guanine derivs. in the (2'-deoxy)ribonucleoside and (2'-deoxy)ribonucleotide pools.

  12. 12

      Kavli, B.; Slupphaug, G.; Krokan, H. Genomic Uracil in Biology, Immunity and Cancer. DNA Damage, DNA Repair and Disease; Royal Society of Chemistry, 2020; Vol. 1, pp 220– 248.

      There is no corresponding record for this reference.

  13. 13

      Krokan, H. E.; Drabløs, F.; Slupphaug, G. Uracil in DNA–Occurrence, Consequences and Repair. Oncogene 2002, 21, 8935– 8948,  DOI: 10.1038/sj.onc.1205996

      13

      Uracil in DNA - occurrence, consequences and repair

      Krokan, Hans E.; Drablos, Finn; Slupphaug, Geir

      Oncogene (2002), 21 (58), 8935-8948CODEN: ONCNES; ISSN:0950-9232. (Nature Publishing Group)

      A review. Uracil in DNA results from deamination of cytosine, resulting in mutagenic U : G mispairs, and misincorporation of dUMP, which gives a less harmful U : A pair. At least four different human DNA glycosylases may remove uracil and thus generate an abasic site, which is itself cytotoxic and potentially mutagenic. These enzymes are UNG, SMUG1, TDG and MBD4. The base excision repair process is completed either by a short patch- or long patch pathway, which largely use different proteins. UNG2 is a major nuclear uracil-DNA glycosylase central in removal of misincorporated dUMP in replication foci, but recent evidence also indicates an important role in repair of U : G mispairs and possibly U in single-stranded DNA. SMUG1 has broader specificity than UNG2 and may serve as a relatively efficient backup for UNG in repair of U : G mismatches and single-stranded DNA. TDG and MBD4 may have specialized roles in the repair of U and T in mismatches in CpG contexts. Recently, a role for UNG2, together with activation induced deaminase (AID) which generates uracil, has been demonstrated in Ig diversification. Studies are now underway to examine whether mice deficient in Ung develop lymphoproliferative malignancies and have a different life span.

  14. 14

      Mundt, J. M.; Hah, S. S.; Sumbad, R. A.; Schramm, V.; Henderson, P. T. Incorporation of Extracellular 8-OxodG into DNA and RNA Requires Purine Nucleoside Phosphorylase in MCF-7 Cells. Nucleic Acids Res. 2007, 36, 228– 236,  DOI: 10.1093/nar/gkm1032

      There is no corresponding record for this reference.

  15. 15

      Hizi, A.; Herzig, E. dUTPase: the Frequently Overlooked Enzyme Encoded by Many Retroviruses. Retrovirology 2015, 12, 70,  DOI: 10.1186/s12977-015-0198-9

      15

      dUTPase: the frequently overlooked enzyme encoded by many retroviruses

      Hizi Amnon; Herzig Eytan

      Retrovirology (2015), 12 (), 70 ISSN:.

      Retroviruses are among the best studied viruses in last decades due to their pivotal involvement in cellular processes and, most importantly, in causing human diseases, most notably-acquired immunodeficiency syndrome (AIDS) that is triggered by human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2, respectively). Numerous studied were conducted to understand the involvement of the three cardinal retroviral enzymes, reverse transcriptase, integrase and protease, in the life cycle of the viruses. These studies have led to the development of many inhibitors of these enzymes as anti-retroviral specific drugs that are used for routine treatments of HIV/AIDS patients. Interestingly, a fourth virus-encoded enzyme, the deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is also found in several major retroviral groups. The presence and the importance of this enzyme to the life cycle of retroviruses were usually overlooked by most retrovirologists, although the occurrence of dUTPases, particularly in beta-retroviruses and in non-primate retroviruses, is known for more than 20 years. Only more recently, retroviral dUTPases were brought into the limelight and were shown in several cases to be essential for viral replication. Therefore, it is likely that future studies on this enzyme will advance our knowledge to a level that will allow designing novel, specific and potent anti-dUTPase drugs that are effective in combating retroviral diseases. The aim of this review is to give concise background information on dUTPases in general and to summarize the most relevant data on retroviral dUTPases and their involvement in the replication processes and pathogenicity of the viruses, as well as in possibly-associated human diseases.

      Gad, H.; Koolmeister, T.; Jemth, A.-S.; Eshtad, S.; Jacques, S. A.; Ström, C. E.; Svensson, L. M.; Schultz, N.; Lundbäck, T.; Einarsdottir, B. O. MTH1 Inhibition Eradicates Cancer by Preventing Sanitation of the dNTP Pool. Nature 2014, 508, 215– 221,  DOI: 10.1038/nature13181

      15

      MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool

      Gad, Helge; Koolmeister, Tobias; Jemth, Ann-Sofie; Eshtad, Saeed; Jacques, Sylvain A.; Stroem, Cecilia E.; Svensson, Linda M.; Schultz, Niklas; Lundbaeck, Thomas; Einarsdottir, Berglind Osk; Saleh, Aljona; Gokturk, Camilla; Baranczewski, Pawel; Svensson, Richard; Berntsson, Ronnie P.-A.; Gustafsson, Robert; Stroemberg, Kia; Sanjiv, Kumar; Jacques-Cordonnier, Marie-Caroline; Desroses, Matthieu; Gustavsson, Anna-Lena; Olofsson, Roger; Johansson, Fredrik; Homan, Evert J.; Loseva, Olga; Braeutigam, Lars; Johansson, Lars; Hoeglund, Andreas; Hagenkort, Anna; Pham, Therese; Altun, Mikael; Gaugaz, Fabienne Z.; Vikingsson, Svante; Evers, Bastiaan; Henriksson, Martin; Vallin, Karl S. A.; Wallner, Olov A.; Hammarstroem, Lars G. J.; Wiita, Elisee; Almloef, Ingrid; Kalderen, Christina; Axelsson, Hanna; Djureinovic, Tatjana; Puigvert, Jordi Carreras; Haeggblad, Maria; Jeppsson, Fredrik; Martens, Ulf; Lundin, Cecilia; Lundgren, Bo; Granelli, Ingrid; Jensen, Annika Jenmalm; Artursson, Per; Nilsson, Jonas A.; Stenmark, Pal; Scobie, Martin; Berglund, Ulrika Warpman; Helleday, Thomas

      Nature (London, United Kingdom) (2014), 508 (7495), 215-221CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)

      Cancers have dysfunctional redox regulation resulting in reactive oxygen species prodn., damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, the authors show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. The authors validate MTH1 as an anticancer target in vivo and describe small mols. TH287 (I) and TH588 (II) as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.

  16. 16

      Wilson, D. L.; Kool, E. T. Ultrafast Oxime Formation Enables Efficient Fluorescence Light-Up Measurement of DNA Base Excision. J. Am. Chem. Soc. 2019, 141, 19379– 19388,  DOI: 10.1021/jacs.9b09812

      16

      Ultrafast Oxime Formation Enables Efficient Fluorescence Light-up Measurement of DNA Base Excision

      Wilson, David L.; Kool, Eric T.

      Journal of the American Chemical Society (2019), 141 (49), 19379-19388CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)

      DNA glycosylases constitute a biol. and biomedically important group of DNA repair enzymes responsible for initiating base excision repair (BER). Measuring their activities can be useful for studying the mechanisms DNA damage and repair and for practical applications in cancer diagnosis and drug screening. Previous fluorescence methods for assaying DNA glycosylases are often complex and/or limited in scope to a single enzyme type. Here we report a universal base excision reporter (UBER) fluorescence probe design that implements an unprecedentedly rapid oxime reaction (>150 M-1 s-1) with high specificity for the abasic (AP) site of DNA. The mol. rotor design achieves a robust >250-500-fold increase in fluorescence upon reaction with AP sites in DNA. By using the fluorescence reporter in concert with specific DNA lesion-contg. substrates, the UBER probe can be used in a coupled assay in principle with any DNA glycosylase. We demonstrate the utility of the UBER probe by assaying five different glycosylases in real time as well as profiling glycosylase activity in cell lysates. We anticipate that the UBER probe will be of considerable utility to researchers studying DNA repair biol. owing to its high level of generalizability, ease of use, and compatibility with biol. derived samples.

  17. 17

      Herbel, W.; Montag, A. Nucleo-Compounds in Protein-Rich Food. Unters. Forsch. 1987, 185, 119– 122,  DOI: 10.1007/BF01850090

      17

      Nucleo-compounds in protein rich food

      Herbel, Walter; Montag, Alfred

      Zeitschrift fuer Lebensmittel-Untersuchung und -Forschung (1987), 185 (2), 119-22CODEN: ZLUFAR; ISSN:0044-3026.

      RNA, DNA, IMP, and pyrimidine and purine bases were detd. in several protein-high foods and the results were tabulated. Heat treatment of the food caused considerable changes in the contents of these substances.

      Lassek, E.; Montag, A. Nucleic Acid Components in Carbohydrate-Rich Food. Unters. Forsch. 1990, 190, 17– 21,  DOI: 10.1007/BF01188257

      17

      Nucleic acid components in carbohydrate-rich food

      Lassek, Eva; Montag, Alfred

      Zeitschrift fuer Lebensmittel-Untersuchung und -Forschung (1990), 190 (1), 17-21CODEN: ZLUFAR; ISSN:0044-3026.

      The nucleic acid component content of numerous foods, esp. those rich in carbohydrates, was investigated. The data obtained for bases (purines and pyrimidines) were calcd. as nucleic acid equiv. (RNA or DNA); the inosine monophosphate content was calcd. from the measured content of hypoxanthine. Not only did cultivated plants such as cereals and pulses show a high RNA-equiv. content but also vegetables such as spinach, leek, broccoli, Chinese cabbage, and cauliflower and mushrooms, including oyster, flat, button and cap mushrooms. In many vegetarian instant meals (principally soups, the presence of autolyzed or hydrolyzed yeast resulted in high purine contents. Most natural foods contg. resting cell tissue, such as grains of seed, exhibit only high-mol.-mass components of various concns.; growing cell tissues (e.g. soybean sprouts) show, however, some low-mol.-mass compds. in addn. to these.

  18. 18

      Lewis, C. A.; Crayle, J.; Zhou, S.; Swanstrom, R.; Wolfenden, R. Cytosine Deamination and the Precipitous Decline of Spontaneous Mutation during Earth’s History. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 8194– 8199,  DOI: 10.1073/pnas.1607580113

      18

      Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history

      Lewis, Charles A. Jr.; Crayle, Jesse; Zhou, Shuntai; Swanstrom, Ronald; Wolfenden, Richard

      Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (29), 8194-8199CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)

      The hydrolytic deamination of cytosine and 5-methylcytosine residues in DNA appears to contribute significantly to the appearance of spontaneous mutations in microorganisms and in human disease. Here, the authors examd. the mechanism of cytosine deamination and the response of the uncatalyzed reaction to changing temp. The pos. charged 1,3-dimethylcytosinium ion was hydrolyzed at a rate similar to the rate of acid-catalyzed hydrolysis of 1-methylcytosine, for which it furnished a satisfactory kinetic model and a probable mechanism. In agreement with earlier reports, uncatalyzed deamination was found to proceed at very similar rates for cytosine, 1-methylcytosine, cytidine, and cytidine 5'-phosphate, and also for cytosine residues in single-stranded DNA generated from a phagemid, in which the authors sequenced an insert representing the gene of HIV-1 virus protease. Arrhenius plots for the uncatalyzed deamination of cytosine were linear over the temp. range of 90-200° and indicated a heat of activation (ΔH⧧) of 23.4 ± 0.5 kcal/mol at pH 7. Recent evidence indicated that the surface of the Earth has been cool enough to support life for >4 × 109 yr and that life has been present for almost as long. If the temp. at Earth's surface is assumed to have followed Newton's law of cooling, declining exponentially from 100 to 25° during that period, then half of the cytosine-deaminating events per unit biomass would have taken place during the 0.2 × 109 yr, and <99.4% would have occurred during the 1st 2 × 109 yr.

  19. 19

      Sahin, S.; Ulusoy, H. I.; Alemdar, S.; Erdogan, S.; Agaoglu, S. The Presence of Polycyclic Aromatic Hydrocarbons (PAHs) in Grilled Beef, Chicken and Fish by Considering Dietary Exposure and Risk Assessment. Food Sci. Anim. Resour. 2020, 40, 675– 688,  DOI: 10.5851/kosfa.2020.e43

      19

      The Presence of Polycyclic Aromatic Hydrocarbons (PAHs) in Grilled Beef, Chicken and Fish by Considering Dietary Exposure and Risk Assessment

      Sahin Seyda; Alemdar Suleyman; Agaoglu Sema; Ulusoy Halil Ibrahim; Erdogan Selim

      Food science of animal resources (2020), 40 (5), 675-688 ISSN:.

      Polycyclic aromatic hydrocarbons (PAHs) are dangerous chemical compounds that can be formed by cooking foods at high temperatures. The aim of this study is to determine the level of contamination of PAH compounds with high performance liquid chromatography (HPLC) on heat treated meat samples and the consumption of PAH compounds in meat samples, as well as the dietary exposure status and possible health risk estimation. In five different heat treated meat samples (meat doner, chicken doner, meatballs, grilled chicken, and fish), the total PAH (Σ16PAH) contamination level was 6.08, 4.42, 4.45, 4.91, and 7.26 μg/kg, respectively. Benzo[a]pyrene (BaP) in meatballs and grilled fish samples had a level 0.70 and 0.73 μg/kg. All of the samples analyzed were found to be below the EU permitted limit (5 μg/kg) in terms of BaP. Estimates of daily intake (EDI) for a total of 16PAH in heat treated meat doner, chicken doner, meatballs, grilled chicken and fish samples were 3.41, 3.71, 2.49, 4.12, and 1.77 ng/kg bw/day, respectively. In this study, the average margin of exposure (MOE) value calculated was found in the range of 179.487 and 425.000 for BaP and PAH4. This study is the first study to provide important information in terms of evaluating the possible health risk that PAH compounds can create in people's diets due to heat treatment of meat and meat products in Sivas, Turkey.

      Sinha, R.; Rothman, N.; Salmon, C.; Knize, M.; Brown, E.; Swanson, C.; Rhodes, D.; Rossi, S.; Felton, J.; Levander, O. Heterocyclic Amine Content in Beef Cooked by Different Methods to Varying Degrees of Doneness and Gravy Made from Meat Drippings. Food Chem. Toxicol. 1998, 36, 279– 287,  DOI: 10.1016/S0278-6915(97)00162-2

      19

      Heterocyclic amine content in beef cooked by different methods to varying degrees of doneness and gravy made from meat drippings

      Sinha, R.; Rothman, N.; Salmon, C. P.; Knize, M. G.; Brown, E. D.; Swanson, C. A.; Rhodes, D.; Rossi, S.; Felton, J. S.; Levander, O. A.

      Food and Chemical Toxicology (1998), 36 (4), 279-287CODEN: FCTOD7; ISSN:0278-6915. (Elsevier Science Ltd.)

      Meats cooked at high temps. sometimes contain heterocyclic amines (HCAs) that are known mutagens and animal carcinogens, but their carcinogenic potential in humans has not been established. To investigate the assocn. between HCAs and cancer, sources of exposure to these compds. need to be detd. Beef is the most frequently consumed meat in the United States and for this study we detd. HCA values in beef samples cooked in ways to represent US cooking practices, the results of which can be used in epidemiol. studies to est. HCA exposure from dietary questionnaires. We measured five HCAs [2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP)] in different types of cooked beef using solid-phase extn. and HPLC. Steak and hamburger patties were pan-fried, oven-broiled, and grilled/barbecued to four levels of doneness (rare, medium, well done or very well done), while beef roasts were oven cooked to three levels of doneness (rare, medium or well done). The measured values of the specific HCAs varied with the cut of beef, cooking method, and doneness level. In general, MeIQx content increased with doneness under each cooking condition for steak and hamburger patties, up to 8.2 ng/g. PhIP was the predominant HCA produced in steak (1.9 to 30 ng/g), but was formed only in very well done fried or grilled hamburger. DiMeIQx was found in trace levels in pan-fried steaks only, while IQ and MeIQ were not detectable in any of the samples. Roast beef did not contain any of the HCAs, but the gravy made from the drippings from well done roasts had 2 ng/g of PhIP and 7 ng/g of MeIQx. Epidemiol. studies need to consider the type of meat, cooking method and degree of doneness/surface browning in survey questions to adequately assess an individual's exposure to HCAs.

  20. 20

      Chatgilialoglu, C.; Ferreri, C.; Geacintov, N. E.; Krokidis, M. G.; Liu, Y.; Masi, A.; Shafirovich, V.; Terzidis, M. A.; Tsegay, P. S. 5′, 8-Cyclopurine Lesions in DNA Damage: Chemical, Analytical, Biological, and Diagnostic Significance. Cells 2019, 8, 513,  DOI: 10.3390/cells8060513

      20

      5',8-cyclopurine lesions in DNA damage: chemical, analytical, biological, and diagnostic significance

      Chatgilialoglu, Chryssostomos; Ferreri, Carla; Geacintov, Nicholas E.; Krokidis, Marios G.; Liu, Yuan; Masi, Annalisa; Shafirovich, Vladimir; Terzidis, Michael A.; Tsegay, Pawlos S.

      Cells (2019), 8 (6), 513CODEN: CELLC6; ISSN:2073-4409. (MDPI AG)

      Purine 5',8-cyclo-2'-deoxynucleosides (cPu) are tandem-type lesions obsd. among the DNA purine modifications and identified in mammalian cellular DNA in vivo. These lesions can be present in two diasteroisomeric forms, 5'R and 5'S, for each 2'-deoxyadenosine and 2'-deoxyguanosine moiety. They are generated exclusively by hydroxyl radical attack to 2'-deoxyribose units generating C5' radicals, followed by cyclization with the C8 position of the purine base. This review describes the main recent achievements in the prepn. of the cPu mol. library for anal. and DNA synthesis applications for the studies of the enzymic recognition and repair mechanisms, their impact on transcription and genetic instability, quant. detn. of the levels of lesions in various types of cells and animal model systems, and relationships between the levels of lesions and human health, disease, and aging, as well as the defining of the detection limits and quantification protocols.

  21. 21

      Zauri, M.; Berridge, G.; Thézénas, M.-L.; Pugh, K. M.; Goldin, R.; Kessler, B. M.; Kriaucionis, S. CDA Directs Metabolism of Epigenetic Nucleosides Revealing a Therapeutic Window in Cancer. Nature 2015, 524, 114– 118,  DOI: 10.1038/nature14948

      21

      CDA directs metabolism of epigenetic nucleosides revealing a therapeutic window in cancer

      Zauri, Melania; Berridge, Georgina; Thezenas, Marie-Laetitia; Pugh, Kathryn M.; Goldin, Robert; Kessler, Benedikt M.; Kriaucionis, Skirmantas

      Nature (London, United Kingdom) (2015), 524 (7563), 114-118CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)

      Cells require nucleotides to support DNA replication and repair damaged DNA. In addn. to de novo synthesis, cells recycle nucleotides from the DNA of dying cells or from cellular material ingested through the diet. Salvaged nucleosides come with the complication that they can contain epigenetic modifications. Because epigenetic inheritance of DNA methylation mainly relies on copying of the modification pattern from parental strands, random incorporation of pre-modified bases during replication could have profound implications for epigenome fidelity and yield adverse cellular phenotypes. Although the salvage mechanism of 5-methyl-2'deoxycytidine (5mdC) has been investigated before, it remains unknown how cells deal with the recently identified oxidized forms of 5mdC: 5-hydroxymethyl-2'deoxycytidine (5hmdC), 5-formy-2'deoxycytidine (5fdC) and 5-carboxyl-2'deoxycytidine (5cadC). Here we show that enzymes of the nucleotide salvage pathway display substrate selectivity, effectively protecting newly synthesized DNA from the incorporation of epigenetically modified forms of cytosine. Thus, cell lines and animals can tolerate high doses of these modified cytidines without any deleterious effects on physiol. Notably, by screening cancer cell lines for growth defects after exposure to 5hmdC, we unexpectedly identify a subset of cell lines in which 5hmdC or 5fdC administration leads to cell lethality. Using genomic approaches, we show that the susceptible cell lines overexpress cytidine deaminase (CDA). CDA converts 5hmdC and 5fdC into variants of uridine that are incorporated into DNA, resulting in accumulation of DNA damage, and ultimately, cell death. Our observations extend current knowledge of the nucleotide salvage pathway by revealing the metab. of oxidized epigenetic bases, and suggest a new therapeutic option for cancers, such as pancreatic cancer, that have CDA overexpression and are resistant to treatment with other cytidine analogs.

      Gratzner, H. G. Monoclonal Antibody to 5-Bromo-and 5-Iododeoxyuridine: A New Reagent for Detection of DNA Replication. Science 1982, 218, 474– 475,  DOI: 10.1126/science.7123245

      21

      Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: a new reagent for detection of DNA replication

      Gratzner, Howard G.

      Science (Washington, DC, United States) (1982), 218 (4571), 474-5CODEN: SCIEAS; ISSN:0036-8075.

      Monoclonal antibodies specific for 5-bromodeoxyuridine have been produced and applied in detecting low levels of DNA replication on a cell-by-cell basis in vitro. The Ig-producing hybridomas were derived from spleen cells of mice immunized with a conjugate of iodouridine and ovalbumin. The cells were fused with the plasmacytoma line SP2/OAg14. The antibodies produced are highly specific for bromodeoxyuridine and iododeoxyuridine and do not crossreact with thymidine. DNA synthesis in cultured cells exposed to bromodeoxyuridine for as short a time as 6 min can be detected easily and rapidly by an immunofluorescent staining method and quantitated by flow cytometry.

      Salic, A.; Mitchison, T. J. A Chemical Method for Fast and Sensitive Detection of DNA Synthesis in vivo. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 2415– 2420,  DOI: 10.1073/pnas.0712168105

      21

      A chemical method for fast and sensitive detection of DNA synthesis in vivo

      Salic, Adrian; Mitchison, Timothy J.

      Proceedings of the National Academy of Sciences of the United States of America (2008), 105 (7), 2415-2420CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)

      We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) and its subsequent detection by a fluorescent azide through a Cu(I)-catalyzed [3 + 2] cycloaddn. reaction ("click" chem.). Detection of the EdU label is highly sensitive and can be accomplished in minutes. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration, allowing the staining of whole-mount prepns. of large tissue and organ explants. In contrast to BrdU, the method does not require sample fixation or DNA denaturation and permits good structural preservation. We demonstrate the use of the method in cultured cells and in the intestine and brain of whole animals.

  22. 22

      Jun, Y. W.; Albarran, E.; Wilson, D. L.; Ding, J.; Kool, E. T. Fluorescence Imaging of Mitochondrial DNA Base Excision Repair Reveals Dynamics of Oxidative Stress Responses. Angew. Chem., Int. Ed. 2022, 61, e202111829,  DOI: 10.1002/anie.202111829

      22

      Fluorescence Imaging of Mitochondrial DNA Base Excision Repair Reveals Dynamics of Oxidative Stress Responses

      Jun, Yong Woong; Albarran, Eddy; Wilson, David L.; Ding, Jun; Kool, Eric T.

      Angewandte Chemie, International Edition (2022), 61 (6), e202111829CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)

      Mitochondrial function in cells declines with aging and with neurodegeneration, due in large part to accumulated mutations in mitochondrial DNA (mtDNA) that arise from deficient DNA repair. However, measuring this repair activity is challenging. We employ a mol. approach for visualizing mitochondrial base excision repair (BER) activity in situ by use of a fluorescent probe (UBER) that reacts rapidly with AP sites resulting from BER activity. Administering the probe to cultured cells revealed signals that were localized to mitochondria, enabling selective observation of mtDNA BER intermediates. The probe showed elevated DNA repair activity under oxidative stress, and responded to suppression of glycosylase activity. Furthermore, the probe illuminated the time lag between the initiation of oxidative stress and the initial step of BER. Absence of MTH1 in cells resulted in elevated demand for BER activity upon extended oxidative stress, while the absence of OGG1 activity limited glycosylation capacity.

  23. 23

      Bonda, E.; Rahav, G.; Kaya, A.; Bakhanashvili, M. p53 in the Mitochondria, as a Trans-Acting Protein, Provides Error-Correction Activities during the Incorporation of Non-Canonical dUTP into DNA. Oncotarget 2016, 7, 73323,  DOI: 10.18632/oncotarget.12331

      23

      p53 in the mitochondria, as a trans-acting protein, provides error-correction activities during the incorporation of non-canonical dUTP into DNA

      Bonda Elad; Rahav Galia; Kaya Angelina; Bakhanashvili Mary; Bakhanashvili Mary

      Oncotarget (2016), 7 (45), 73323-73336 ISSN:.

      Mutations in mitochondrial DNA is an outcome of errors produced by DNA polymerase γ during replication and failure of the repair mechanism. Misincorporation of non-canonical dUTP leads to mutagenesis or apoptosis, and may contribute to the cytotoxic effects of 5'-fluorouracil chemotherapy. Tumor suppressor p53 protein in the mitochondria displays physical and functional interactions with mitochondrial DNA and polymerase γ, and by its intrinsic 3'→5' exonuclease activity can diminish the polymerization errors. Here we demonstrate the impact of p53 on incorporation of uracil into DNA examined with mitochondrial fractions, as the source of polymerase γ. p53 in mitochondria facilitates DNA damage repair functions resulting from uracil-DNA misincorporation. Our biochemical studies revealed that the procession of U:A and mismatched U:G lesions enhances in the presence of recombinant or endogenous cytoplasmic p53. p53 in mitochondria can function as an exonuclease/proofreader for polymerase γ by either decreasing the incorporation of non-canonical dUTP into DNA or by promoting the excision of incorporated nucleotide from nascent DNA, thus expanding the spectrum of DNA damage sites exploited for proofreading as a trans-acting protein. The data suggest that p53 may contribute to defense of the cells from consequences of dUTP misincorporation in both normal and tumor cells.

  24. 24

      Yano, W.; Yokogawa, T.; Wakasa, T.; Yamamura, K.; Fujioka, A.; Yoshisue, K.; Matsushima, E.; Miyahara, S.; Miyakoshi, H.; Taguchi, J. TAS-114, a First-in-Class Dual dUTPase/DPD Inhibitor, Demonstrates Potential to Improve Therapeutic Efficacy of Fluoropyrimidine-Based Chemotherapy. Mol. Cancer Ther. 2018, 17, 1683– 1693,  DOI: 10.1158/1535-7163.MCT-17-0911

      24

      TAS-114, a First-in-Class Dual dUTPase/DPD Inhibitor, Demonstrates Potential to Improve Therapeutic Efficacy of Fluoropyrimidine-Based Chemotherapy

      Yano, Wakako; Yokogawa, Tatsushi; Wakasa, Takeshi; Yamamura, Keisuke; Fujioka, Akio; Yoshisue, Kunihiro; Matsushima, Eiji; Miyahara, Seiji; Miyakoshi, Hitoshi; Taguchi, Junko; Chong, Khoon Tee; Takao, Yayoi; Fukuoka, Masayoshi; Matsuo, Kenichi

      Molecular Cancer Therapeutics (2018), 17 (8), 1683-1693CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)

      In this study, we designed a novel small mol. inhibitor, TAS-114, which targets the intercellular metab. of 5-FU to enhance antitumor activity and modulates catabolic pathway to improve the systemic availability of 5-FU. TAS-114 strongly and competitively inhibited deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), a gatekeeper protein preventing aberrant base incorporation into DNA, and enhanced the cytotoxicity of fluoropyrimidines in cancer cells; however, it had little intrinsic activity. In addn., TAS-114 had moderate and reversible inhibitory activity on dihydropyrimidine dehydrogenase (DPD), a catabolizing enzyme of 5-FU. Thus, TAS-114 increased the bioavailability of 5-FU when coadministered with capecitabine in mice, and it significantly improved the therapeutic efficacy of capecitabine by reducing the required dose of the prodrug by dual enzyme inhibition. Enhancement of antitumor efficacy caused by the addn. of TAS-114 was retained in the presence of a potent DPD inhibitor contg. oral fluoropyrimidine (S-1), indicating that dUTPase inhibition plays a major role in enhancing the antitumor efficacy of fluoropyrimidine-based therapy. In conclusion, TAS-114, a dual dUTPase/DPD inhibitor, demonstrated the potential to improve the therapeutic efficacy of fluoropyrimidine. Dual inhibition of dUTPase and DPD is a novel strategy for the advancement of oral fluoropyrimidine-based chemotherapy for cancer treatment.

  25. 25

      Ladner, R. D. The Role of dUTPase and Uracil-DNA Repair in Cancer Chemotherapy. Curr. Protein Pept. Sci. 2001, 2, 361– 370,  DOI: 10.2174/1389203013380991

      25

      The role of dUTPase and uracil-DNA repair in cancer chemotherapy

      Ladner, Robert D.

      Current Protein and Peptide Science (2001), 2 (4), 361-370CODEN: CPPSCM; ISSN:1389-2037. (Bentham Science Publishers Ltd.)

      A review with refs. Thymidylate metab. is an important target for chemotherapeutic agents that combat a variety of neoplastic diseases including head and neck, breast and gastrointestinal cancers. Therapeutic strategies applied to this pathway target the thymidylate synthase (TS) reaction that catalyzes the reductive methylation of deoxyuridylate (dUMP) to form thymidylate (TMP). This reaction represents the sole de novo source of TMP required for DNA replication and repair. Inhibitors of this pathway include the widely utilized fluoropyrimide and antifolate classes of anti-cancer agents. Studies attempting to elucidate the mol. mechanisms of cell killing mediated by inhibitors of the TS reaction suggest that cytotoxicity results from a process known as "thymineless death". This term describes the extreme TTP pool depletion obsd. following TS inhibition. Although depletion of TTP pools is clearly involved in this process, there is now considerable evidence implicating aberrant uracil-DNA metab. as an important mechanism of toxicity. Upon TS inhibition, dUTP pools may accumulate, inducing repeated cycles of uracil misincorporation into DNA and repair-mediated DNA damage. Central to the uracil-misincorporation pathway are the enzymes deoxyuridine nucleotidohydrolase (dUTPase) (EC 3.6.1.23) and uracil-DNA glycosylase (UDG) (EC 3.2.2.3). DUTPase catalyzes the hydrolysis of dUTP to form dUMP and pyrophosphate thereby eliminating dUTP and preventing its utilization by DNA polymerases during replication and repair. UDG initiates the base excision repair pathway effectively removing any uracil residues that may arise in DNA. Under normal conditions, uracil is precluded from DNA by the combined actions of dUTPase and UDG. However, during TS inhibition, dUTP pools may accumulate and overwhelm dUTPase, resulting in repeated cycles of uracil misincorporation and detrimental repair leading to stand breaks and cell death. Because dUTPase plays a pivotal role in regulating cellular dUTP pools, this enzyme could have profound effects on the efficacy of agents that target thymidylate biosynthesis. This article reviews our current understanding of the role of aberrant uracil-DNA metab. as a contributing mechanism of cytotoxicity initiated by chemotherapeutic agents that target de novo thymidylate metab. The role of dUTPase expression in modulating therapeutic response is presented including evidence from yeast and mammalian cell culture models and clin. studies. The regulation of human dUTPase isoforms in normal and neoplastic tissues will be reviewed as well as the role of dUTPase expression as a prognostic marker for overall survival and response to therapy in colon cancer.

  26. 26

      Cannan, W. J.; Pederson, D. S. Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin. J. Cell. Physiol. 2016, 231, 3– 14,  DOI: 10.1002/jcp.25048

      26

      Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin

      Cannan, Wendy J.; Pederson, David S.

      Journal of Cellular Physiology (2016), 231 (1), 3-14CODEN: JCLLAX; ISSN:0021-9541. (Wiley-Blackwell)

      A review. All organisms suffer double-strand breaks (DSBs) in their DNA as a result of exposure to ionizing radiation. DSBs can also form when replication forks encounter DNA lesions or repair intermediates. The processing and repair of DSBs can lead to mutations, loss of heterozygosity, and chromosome rearrangements that result in cell death or cancer. The most common pathway used to repair DSBs in metazoans (non-homologous DNA end joining) is more commonly mutagenic than the alternative pathway (homologous recombination mediated repair). Thus, factors that influence the choice of pathways used DSB repair can affect an individual's mutation burden and risk of cancer. This review describes radiol., chem., and biol. mechanisms that generate DSBs, and discusses the impact of such variables as DSB etiol., cell type, cell cycle, and chromatin structure on the yield, distribution, and processing of DSBs. The final section focuses on nucleosome-specific mechanisms that influence DSB prodn., and the possible relationship between higher order chromosome coiling and chromosome shattering (chromothripsis).

  27. 27

      Eccles, L. J.; O’Neill, P.; Lomax, M. E. Delayed Repair of Radiation Induced Clustered DNA Damage: Friend or Foe?. Mutat. Res. - Fundam. Mol. Mec. 2011, 711, 134– 141,  DOI: 10.1016/j.mrfmmm.2010.11.003

      27

      Delayed repair of radiation induced clustered DNA damage: Friend or foe?

      Eccles, Laura J.; O'Neill, Peter; Lomax, Martine E.

      Mutation Research, Fundamental and Molecular Mechanisms of Mutagenesis (2011), 711 (1-2), 134-141CODEN: MUREAV; ISSN:0027-5107. (Elsevier B.V.)

      A review. A signature of ionizing radiation exposure is the induction of DNA clustered damaged sites, defined as two or more lesions within one to two helical turns of DNA by passage of a single radiation track. Clustered damage is made up of double strand breaks (DSB) with assocd. base lesions or abasic (AP) sites, and non-DSB clusters comprised of base lesions, AP sites and single strand breaks. This review will conc. on the exptl. findings of the processing of non-DSB clustered damaged sites. It has been shown that non-DSB clustered damaged sites compromise the base excision repair pathway leading to the lifetime extension of the lesions within the cluster, compared to isolated lesions, thus the likelihood that the lesions persist to replication and induce mutation is increased. In addn. certain non-DSB clustered damaged sites are processed within the cell to form addnl. DSB. The use of E. coli to demonstrate that clustering of DNA lesions is the major cause of the detrimental consequences of ionizing radiation is also discussed. The delayed repair of non-DSB clustered damaged sites in humans can be seen as a "friend", leading to cell killing in tumor cells or as a "foe", resulting in the formation of mutations and genetic instability in normal tissue.

      Thompson, P. S.; Cortez, D. New Insights into Abasic Site Repair and Tolerance. DNA repair 2020, 90, 102866,  DOI: 10.1016/j.dnarep.2020.102866

      27

      New insights into abasic site repair and tolerance

      Thompson, Petria S.; Cortez, David

      DNA Repair (2020), 90 (), 102866CODEN: DRNEAR; ISSN:1568-7864. (Elsevier B.V.)

      A review. Thousands of apurinic/apyrimidinic (AP or abasic) sites form in each cell, each day. This simple DNA lesion can have profound consequences to cellular function, genome stability, and disease. As potent blocks to polymerases, they interfere with the reading and copying of the genome. Since they provide no coding information, they are potent sources of mutation. Due to their reactive chem., they are intermediates in the formation of lesions that are more challenging to repair including double-strand breaks, interstrand crosslinks, and DNA protein crosslinks. Given their prevalence and deleterious consequences, cells have multiple mechanisms of repairing and tolerating these lesions. While base excision repair of abasic sites in double-strand DNA has been studied for decades, new interest in abasic site processing has come from more recent insights into how they are processed in single-strand DNA. In this review, we discuss the source of abasic sites, their biol. consequences, tolerance mechanisms, and how they are repaired in double and single-stranded DNA.

  28. 28

      Mah, L.; El-Osta, A.; Karagiannis, T. γH2AX: A Sensitive Molecular Marker of DNA Damage and Repair. Leukemia 2010, 24, 679– 686,  DOI: 10.1038/leu.2010.6

      28

      γH2AX: a sensitive molecular marker of DNA damage and repair

      Mah, L.-J.; El-Osta, A.; Karagiannis, T. C.

      Leukemia (2010), 24 (4), 679-686CODEN: LEUKED; ISSN:0887-6924. (Nature Publishing Group)

      A review. Phosphorylation of the Ser-139 residue of the histone variant H2AX, forming γH2AX, is an early cellular response to the induction of DNA double-strand breaks. Detection of this phosphorylation event has emerged as a highly specific and sensitive mol. marker for monitoring DNA damage initiation and resoln. Further, anal. of γH2AX foci has numerous other applications including, but not limited to, cancer and aging research. Quantitation of γH2AX foci has also been applied as a useful tool for the evaluation of the efficacy of various developmental drugs, particularly, radiation modifying compds. This review focuses on the current status of γH2AX as a marker of DNA damage and repair in the context of ionizing radiation. Although the emphasis is on γ-radiation-induced γH2AX foci, the effects of other genotoxic insults including exposure to UV rays, oxidative stress and chem. agents are also discussed.

  29. 29

      Haskins, J. S.; Su, C.; Maeda, J.; Walsh, K. D.; Haskins, A. H.; Allum, A. J.; Froning, C. E.; Kato, T. A. Evaluating the Genotoxic and Cytotoxic Effects of Thymidine Analogs, 5-Ethynyl-2′-Deoxyuridine and 5-Bromo-2′-Deoxyurdine to Mammalian Cells. Int. J. Mol. Sci. 2020, 21, 6631,  DOI: 10.3390/ijms21186631

      29

      Evaluating the genotoxic and cytotoxic effects of thymidine analogs, 5-ethynyl-2-deoxyuridine and 5-bromo-2-deoxyurdine to mammalian cells

      Haskins, Jeremy S.; Su, Cathy; Maeda, Junko; Walsh, Kade D.; Haskins, Alexis H.; Allum, Allison J.; Froning, Coral E.; Kato, Takamitsu A.

      International Journal of Molecular Sciences (2020), 21 (18), 6631CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)

      BrdU (bromodeoxyuridine) and EdU (ethynyldeoxyuridine) have been largely utilized as the means of monitoring DNA replication and cellular division. Although BrdU induces gene and chromosomal mutations and induces sensitization to photons, EdU's effects have not been extensively studied yet. Therefore, we investigated EdU's potential cytotoxic and mutagenic effects and its related underlying mechanisms when administered to Chinese hamster ovary (CHO) wild type and DNA repair-deficient cells. EdU treatment displayed a higher cytotoxicity and genotoxicity than BrdU treatment. Cells with defective homologous recombination repair displayed a greater growth delay and severe inhibition of clonogenicity with EdU compared to wild type and other DNA repair-deficient cells. Inductions of sister chromatid exchange and hypoxanthine phosphorybosyl transferase (HPRT) mutation were obsd. in EdU-incorporated cells as well. Interestingly, on the other hand, EdU did not induce sensitization to photons to the same degree as BrdU. Our results demonstrate that elevated concns. (similar to manufacturers suggested concn.; >5-10μM) of EdU treatment were toxic to the cell cultures, particularly in cells with a defect in homologous recombination repair. Therefore, EdU should be administered with addnl. precautions.

  30. 30

      LeBlanc, D. P.; Meier, M.; Lo, F. Y.; Schmidt, E.; Valentine, C.; Williams, A.; Salk, J. J.; Yauk, C. L.; Marchetti, F. Duplex Sequencing Identifies Genomic Features that Determine Susceptibility to Benzo[a]pyrene-Induced in vivo Mutations. BMC Genomics 2022, 23, 542,  DOI: 10.1186/s12864-022-08752-w

      30

      Duplex sequencing identifies genomic features that determine susceptibility to benzo(a)pyrene-induced in vivo mutations

      LeBlanc, Danielle P. M.; Meier, Matthew; Lo, Fang Yin; Schmidt, Elizabeth; Valentine III, Charles; Williams, Andrew; Salk, Jesse J.; Yauk, Carole L.; Marchetti, Francesco

      BMC Genomics (2022), 23 (1), 542CODEN: BGMEET; ISSN:1471-2164. (BioMed Central Ltd.)

      Exposure to environmental mutagens increases the risk of cancer and genetic disorders. We used Duplex Sequencing (DS), a high-accuracy error-cor. sequencing technol., to analyze mutation induction across twenty 2.4 kb intergenic and genic targets in the bone marrow of MutaMouse males exposed to benzo(a)pyrene (BaP), a widespread environmental pollutant. DS revealed a linear dose-related induction of mutations across all targets with low intra-group variability. Heterochromatic and intergenic regions exhibited the highest mutation frequencies (MF). C:G > A:T transversions at CCA, CCC and GCC trinucleotides were enriched in BaP-exposed mice consistent with the known etiol. of BaP mutagenesis. However, GC-content had no effect on mutation susceptibility. A pos. correlation was obsd. between DS and the "gold-std." transgenic rodent gene mutation assay. Overall, we demonstrate that DS is a promising approach to study in vivo mutagenesis and yields crit. insight into the genomic features governing mutation susceptibility, spectrum, and variability across the genome.

  31. 31

      Theisinger, A.; Grenacher, B.; Rech, K.; Scharrer, E. Nucleosides are Efficiently Absorbed by Na-Dependent Transport Across the Intestinal Brush Border Membrane in Veal Calves. J. Dairy Sci. 2002, 85, 2308– 2314,  DOI: 10.3168/jds.S0022-0302(02)74311-7

      31

      Nucleosides are efficiently absorbed by Na+-dependent transport across the intestinal brush border membrane in veal calves

      Theisinger, Anja; Grenacher, B.; Rech, K. S.; Scharrer, E.

      Journal of Dairy Science (2002), 85 (9), 2308-2314CODEN: JDSCAE; ISSN:0022-0302. (American Dairy Science Association)

      In previous work, a comparatively high capacity for Na+-dependent transport of nucleosides across the intestinal brush border membrane (BBM) was obsd. in dairy cows, which might be related to digestion of the large amt. of nucleic acids present in ruminal microorganisms in the ruminant small intestine. If this were the case, the capacity for Na+-dependent intestinal nucleoside transport should be much lower in veal calves, in which only small amts. of nucleic acids, nucleotides, and nucleosides reach the small intestine via the milk replacer. To test this hypothesis, we investigated Na+-dependent transport of 3H-labeled thymidine and guanosine across the BBM using BBM vesicles (BBMV) isolated from the small intestine of veal calves. In the presence of a transmembrane Na+ gradient both substrates were transported against a concn. gradient. Inhibitory studies showed that thymidine and guanosine are transported by two different transporters with overlapping substrate specificity, one accepting predominantly pyrimidine nucleosides (N2) and one accepting particularly purine nucleosides (N1). Nucleoside transport was inhibited by glucose along the whole small intestine. Maximal transport rates similar to those in dairy cows were obtained for the proximal, mid-, and distal small intestine. These findings suggest that the high absorptive capacity for nucleosides is a genetically fixed property in the bovine small intestine, which is already present in the preruminant state of veal calves. It may contribute to the high digestibility of nucleic acids obsd. by others in veal calves receiving milk replacer supplemented with RNA. Its main function may be the efficient absorption of nucleosides resulting from the digestion of nucleic acids assocd. with desquamated enterocytes. Due to the limited de novo synthesis of nucleotides in enterocytes intracellular uptake of nucleosides across the BBM may contribute to nucleic acid synthesis in enterocytes and thus may have a trophic effect on the intestinal epithelium.

  32. 32

      Cadet, J.; Wagner, J. R. DNA Base Damage by Reactive Oxygen Species, Oxidizing Agents, and UV Radiation. Cold Spring Harb. Perspect. Biol. 2013, 5, a012559,  DOI: 10.1101/cshperspect.a012559

      32

      DNA base damage by reactive oxygen species, oxidizing agents, and UV radiation

      Cadet, Jean; Wagner, J. Richard

      Cold Spring Harbor Perspectives in Biology (2013), 5 (2), A012559/1-A012559/18CODEN: CSHPEU; ISSN:1943-0264. (Cold Spring Harbor Laboratory Press)

      A review. Emphasis has been placed in this article dedicated to DNA damage on recent aspects of the formation and measurement of oxidatively generated damage in cellular DNA in order to provide a comprehensive and updated survey. This includes single pyrimidine and purine base lesions, intrastrand crosslinks, purine 5',8-cyclonucleosides, DNA-protein adducts and interstrand crosslinks formed by the reactions of either the nucleobases or the 2-deoxyribose moiety with the hydroxyl radical, one-electron oxidants, singlet oxygen, and hypochlorous acid. In addn., recent information concerning the mechanisms of formation, individual measurement, and repair-rate assessment of bipyrimidine photoproducts in isolated cells and human skin upon exposure to UVB radiation, UVA photons, or solar simulated light is critically reviewed.

  33. 33

      Chen, Y.; Liu, X.; Sun, X.; Zhang, J.; Mi, Y.; Li, Q.; Guo, Z. Synthesis and Antioxidant Activity of Cationic 1, 2, 3-Triazole Functionalized Starch Derivatives. Polymers 2020, 12, 112,  DOI: 10.3390/polym12010112

      33

      Synthesis and antioxidant activity of cationic 1,2,3-triazole functionalized starch derivatives

      Chen, Yuan; Liu, Xiguang; Sun, Xueqi; Zhang, Jingjing; Mi, Yingqi; Li, Qing; Guo, Zhanyong

      Polymers (Basel, Switzerland) (2020), 12 (1), 112CODEN: POLYCK; ISSN:2073-4360. (MDPI AG)

      In this study, starch was chem. modified to improve its antioxidant activity. Five novel cationic 1,2,3-triazole functionalized starch derivs. were synthesized by using "click" reaction and N-alkylation. A convenient method for pre-azidation of starch was developed. The structures of the derivs. were analyzed using FTIR and 1H NMR. The radicals scavenging abilities of the derivs. against hydroxyl radicals, DPPH radicals, and superoxide radicals were tested in vitro in order to evaluate their antioxidant activity. Results revealed that all the cationic starch derivs. (2a-2e), as well as the precursor starch derivs. (1a-1e), had significantly improved antioxidant activity compared to native starch. In particular, the scavenging ability of the derivs. against superoxide radicals was extremely strong. The improved antioxidant activity benefited from the enhanced soly. and the added pos. charges. The biocompatibility of the cationic derivs. was confirmed by the low hemolytic rate (<2%). The obtained derivs. in this study have great potential as antioxidant materials that can be applied in the fields of food and biomedicine.

      Yang, S.; Guo, Z.; Miao, F.; Xue, Q.; Qin, S. The Hydroxyl Radical Scavenging Activity of Chitosan, Hyaluronan, Starch and Their O-Carboxymethylated Derivatives. Carbohydr. Polym. 2010, 82, 1043– 1045,  DOI: 10.1016/j.carbpol.2010.06.014

      33

      The hydroxyl radical scavenging activity of chitosan, hyaluronan, starch and their O-carboxymethylated derivatives

      Yang, Shaoli; Guo, Zhanyong; Miao, Fengping; Xue, Qinzhao; Qin, Song

      Carbohydrate Polymers (2010), 82 (4), 1043-1045CODEN: CAPOD8; ISSN:0144-8617. (Elsevier Ltd.)

      In order to study the effect of active hydroxyl and amino groups on scavenging activity against hydroxyl radicals of polysaccharides, three kinds of carboxymethylated polysaccharides (carboxymethyl chitosan (O-CM-chitosan), carboxymethyl hyaluronan (CMHA), and carboxymethyl starch (CMS)) were prepd. and their antioxidant activities against hydroxyl radicals were assessed, resp. Results showed that O-CM-chitosan had lower scavenging ability on hydroxyl radicals than chitosan. CMHA and CMS had the same tendency. For the three kinds of polysaccharides, scavenging ability on hydroxyl radicals was found to be in the order of chitosan > HA > starch. The scavenging ability of carboxymethylated polysaccharides had the same order as related to its corresponding polysaccharides at higher concns. (≥0.8 mg/mL). There were not only hydroxyl groups but also amino or acetamino (CH3CONH-) groups in the mols. of chitosan and HA, but only hydroxyl group for starch. It was suggested that the sequence influence the scavenging activity against hydroxyl radicals might be amino group > acetamide group > hydroxyl group.

  34. 34

      Watling, C. Z.; Schmidt, J. A.; Dunneram, Y.; Tong, T. Y.; Kelly, R. K.; Knuppel, A.; Travis, R. C.; Key, T. J.; Perez-Cornago, A. Risk of Cancer in Regular and Low Meat-Eaters, Fish-Eaters, and Vegetarians: a Prospective Analysis of UK Biobank Participants. BMC Medicine 2022, 20, 73,  DOI: 10.1186/s12916-022-02256-w

      34

      Risk of cancer in regular and low meat-eaters, fish-eaters, and vegetarians: a prospective analysis of UK Biobank participants

      Watling Cody Z; Schmidt Julie A; Dunneram Yashvee; Tong Tammy Y N; Kelly Rebecca K; Knuppel Anika; Travis Ruth C; Key Timothy J; Perez-Cornago Aurora

      BMC medicine (2022), 20 (1), 73 ISSN:.

      BACKGROUND: Following a vegetarian diet has become increasingly popular and some evidence suggests that being vegetarian may be associated with a lower risk of cancer overall. However, for specific cancer sites, the evidence is limited. Our aim was to assess the associations of vegetarian and non-vegetarian diets with risks of all cancer, colorectal cancer, postmenopausal breast cancer, and prostate cancer and to explore the role of potential mediators between these associations. METHODS: We conducted a prospective analysis of 472,377 UK Biobank participants who were free from cancer at recruitment. Participants were categorised into regular meat-eaters (n = 247,571), low meat-eaters (n = 205,385), fish-eaters (n = 10,696), and vegetarians (n = 8685) based on dietary questions completed at recruitment. Multivariable-adjusted Cox regressions were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for all cancer incidence and separate cancer sites across diet groups. RESULTS: After an average follow-up of 11.4 years, 54,961 incident cancers were identified, including 5882 colorectal, 7537 postmenopausal breast, and 9501 prostate cancers. Compared with regular meat-eaters, being a low meat-eater, fish-eater, or vegetarian were all associated with a lower risk of all cancer (HR: 0.98, 95% CI: 0.96-1.00; 0.90, 0.84-0.96; 0.86, 0.80-0.93, respectively). Being a low meat-eater was associated with a lower risk of colorectal cancer in comparison to regular meat-eaters (0.91, 0.86-0.96); however, there was heterogeneity in this association by sex (p = 0.007), with an inverse association across diet groups in men, but not in women. Vegetarian postmenopausal women had a lower risk of breast cancer (0.82, 0.68-0.99), which was attenuated and non-significant after adjusting for body mass index (BMI; 0.87, 0.72-1.05); in mediation analyses, BMI was found to possibly mediate the observed association. In men, being a fish-eater or a vegetarian was associated with a lower risk of prostate cancer (0.80, 0.65-0.99 and 0.69, 0.54-0.89, respectively). CONCLUSION: The lower risk of colorectal cancer in low meat-eaters is consistent with previous evidence suggesting an adverse impact of meat intake. The lower risk of postmenopausal breast cancer in vegetarian women may be explained by their lower BMI. It is not clear whether the other differences observed for all cancers and for prostate cancer reflect any causal relationships or are due to other factors such as residual confounding or differences in cancer detection.

  35. 35

      Nagy, G. N.; Leveles, I.; Vértessy, B. G. Preventive DNA Repair by Sanitizing the Cellular (Deoxy) Nucleoside Triphosphate Pool. FEBS journal 2014, 281, 4207– 4223,  DOI: 10.1111/febs.12941

      35

      Preventive DNA repair by sanitizing the cellular (deoxy)nucleoside triphosphate pool

      Nagy, Gergely N.; Leveles, Ibolya; Vertessy, Beata G.

      FEBS Journal (2014), 281 (18), 4207-4223CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)

      A review. The occurrence of modified bases in DNA is attributed to some major factors: incorporation of altered nucleotide building blocks and chem. reactions or radiation effects on bases within the DNA structure. Several 'house-cleaning' enzyme families are involved in preventing the incorporation of noncanonical bases playing a 'sanitizing' role. The catalytic mechanism of action of these enzymes has been revealed for a no. of representatives in clear structural and kinetic detail. Here, the authors focus in detail on those examples where clear evidence has been produced using high-resoln. structural studies. Comparing the protein fold and architecture of the enzyme active sites, 2 main classes of sanitizing deoxyribonucleoside triphosphate pyrophosphatases can be assigned that are distinguished by the site of nucleophilic attack. In enzymes assocd. with attack at the α-P atom, it is shown that coordination of the γ-phosphate group is also ensured by multiple interactions. By contrast, enzymes catalyzing attack at the β-P atom mainly coordinate the α- and the β-phosphate only. Characteristic differences are also obsd. with respect to the role of the metal ion cofactor (Mg2+) and the coordination of nucleophilic water. Using different catalytic mechanisms embedded in different protein folds, these enzymes present a clear example of convergent evolution.

  36. 36

      Kiwerska, K.; Szyfter, K. DNA Repair in Cancer Initiation, Progression, and Therapy─a Double-Edged Sword. J. Appl. Genet. 2019, 60, 329– 334,  DOI: 10.1007/s13353-019-00516-9

      36

      DNA repair in cancer initiation, progression, and therapy-a double-edged sword

      Kiwerska, Katarzyna; Szyfter, Krzysztof

      Journal of Applied Genetics (2019), 60 (3-4), 329-334CODEN: JAGEFC; ISSN:1234-1983. (Springer GmbH)

      Genomic and mitochondrial DNA mols. are exposed continuously for a damaging activity of chem., phys., and internal genotoxicants. When DNA repair machinery is not working efficiently, the generation of DNA lesions and mutations leads to carcinogenic transformation. The high no. of mutation going up to 105 per cell was recognized as a driving force of oncogenesis. Moreover, a high activity of DNA repair genes was hypothesized as a predisposition to metastasis. DNA repair potential has to be taken into account attempting to chemo- and/or radiotherapy. A low activity of DNA repair genes makes tumor cells more sensitive to therapy, but on the other hand, non-tumor cells getting lesions could form second primary cancer. Contrary, high activity of DNA repair genes counteracts attempted therapy. It means an individualized therapy based on recognition of DNA repair potential is recommended.

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