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Abstract: The human buccal micronucleus cytome assay (BMCyt) is one of the most widely used techniques to measure genetic damage in human population studies. Reducing protocol variability, assessing the role of confounders, and estimating a range of reference values are research priorities that will be addressed by the HUMNXL collaborative study. The HUMNXL project evaluates the impact of host factors, occupation, life-style, disease status, and protocol features on the occurrence of MN in exfoliated buccal cells. In addition, the study will provide a range of reference values for all cytome endpoints. A database of 5424 subjects with buccal MN values obtained from 30 laboratories worldwide was compiled and analyzed to investigate the influence of several conditions affecting MN frequency. Random effects models were mostly used to investigate MN predictors. The estimated spontaneous MN frequency was 0.74‰ (95% CI 0.52–1.05). Only staining among technical features influenced MN frequency, with an abnormal increase for non-DNA-specific stains. No effect of gender was evident, while the trend for age was highly significant (p <0.001). Most occupational exposures and a diagnosis of cancer significantly increased MN and other endpoints frequencies. MN frequency increased in heavy smoking (≥40cig/day, FR=1.37; 95% CI 1.03–.82) and decreased with daily fruit consumption (FR=0.68; 95% CI 0.50–0.91). The results of the HUMNXL project identified priorities for validation studies, increased the basic knowledge of the assay, and contributed to the creation of a laboratory network which in perspective may allow the evaluation of disease risk associated with MN frequency. [Copyright &y& Elsevier]

Aim: To quantitatively evaluate the inhibition of human DNA repair proteins APE1 and MTH1 by dextran-coated γ-Fe 2 O 3 ultrasmall superparamagnetic iron oxide nanoparticles (dUSPIONs). Materials & methods : Liquid chromatography-tandem mass spectrometry with isotope-dilution was used to measure the expression levels of APE1 and MTH1 in MCL-5 cells exposed to increasing doses of dUSPIONs. The expression levels of APE1 and MTH1 were measured in cytoplasmic and nuclear fractions of cell extracts. Results: APE1 and MTH1 expression was significantly inhibited in both cell fractions at the highest dUSPION dose. The expression of MTH1 was linearly inhibited across the full dUSPION dose range in both fractions. Conclusion: These findings warrant further studies to characterize the capacity of dUSPIONs to inhibit other DNA repair proteins in vitro and in vivo .

Dietary exposure to aflatoxin B 1 (AFB 1 ) is a significant contributor to the incidence of hepatocellular carcinomas globally. AFB 1 exposure leads to the formation of AFB 1 -N 7 -guanine (AFB 1 -N 7 -Gua) and two diastereomers of the imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B 1 (AFB 1 -FapyGua) in DNA. These adducts lead to G → T transversion mutations with the ring-opened adduct being more mutagenic than the cationic species. Accurate measurement of these three adducts as biomarkers in DNA and urine will help identify dietary exposure to AFB 1 as a risk factor in the development of hepatocellular carcinoma worldwide. Herein, we report an improved methodology for the measurement of AFB 1 -N 7 -Gua and the two diastereomers of AFB 1 -FapyGua using liquid chromatography-tandem mass spectrometry with isotope dilution. We measured the levels of these compounds in liver DNA of six control mice and six AFB 1 -treated mice. Levels varying from 1.5 to 45 lesions/10 6 DNA bases in AFB 1 -treated mice were detected depending on the compound and animal. No background levels of these adducts were detected in control mice. We also tested whether the AFB 1 treatment caused oxidatively induced DNA base damage using gas chromatography-tandem mass spectrometry with isotope dilution. Although background levels of several pyrimidine- and purine-derived lesions were detected, no increases in these levels were found upon AFB 1 treatment of mice. On the other hand, significantly increased levels of (5' R)- and (5' S)-8,5'-cyclo-2'-deoxyadenosines were observed in liver DNA of AFB 1 -treated mice. The impact of this work is expected to achieve the accurate measurement of three AFB 1 -DNA adducts and oxidatively induced DNA lesions as biomarkers of AFB 1 exposure as germane to investigations designed for the prevention of aflatoxin-related hepatocellular carcinomas and for determining the effects of genetic deficiencies in human populations.

Background and Objectives: The aim of this study was to compare the effects of sevoflurane and propofol anesthesia on oxidative DNA damage that occurs in low-extremity ischemia and is caused by tourniquet application., Methods: Fourteen New Zealand rabbits were randomly allocated into two equal groups. Group S (n=7) received sevoflurane (2.5-4 percent) inhalation and Group P (n=7) received a propofol infusion (1-2mg·kg -1 ·min -1 ), after which a pneumatic tourniquet was placed on the right lower extremity. Blood samples were collected prior to tourniquet placement (baseline), 120min after ischemia, 15min after ischemia and 120minutes (min) after ischemia. Malondialdehyde (MDA) levels were analyzed to determine lipid peroxidation, and single cell gel electrophoresis (SCGE) was used to determine DNA damage., Results: At 15min after ischemia, the MDA levels in Group P (8.15±2.61μM) were higher than baseline (6.26±3.19μM, p=0.026) and Group S (4.98±0.77μM, p=0.01). DNA damage was similar in both groups, although DNA damage was higher than baseline (tail moment 0.63±0.27, tail intensity 3.76±1.26) in Group P at the 15th minute of reperfusion (tail moment 1.05±0.45, p=0.06; tail intensity 5.33±1.56, p=0.01). The increase in tail moment and tail intensity returned to normal levels in both groups 2hours after the termination of ischemia., Conclusion: Given that oxidative stress and genotoxic effect disappear in the late stages of reperfusion, we conclude that neither sevoflurane nor propofol can be considered superior to the other in anesthesia practices for extremity surgeries involving the use of a tourniquet., (Copyright © 2016. Publicado por Elsevier Editora Ltda.)

Endogenous and exogenous reactive species cause oxidatively induced DNA damage in living organisms by a variety of mechanisms. As a result, a plethora of mutagenic and/or cytotoxic products are formed in cellular DNA. This type of DNA damage is repaired by base excision repair, although nucleotide excision repair also plays a limited role. DNA glycosylases remove modified DNA bases from DNA by hydrolyzing the glycosidic bond leaving behind an apurinic/apyrimidinic (AP) site. Some of them also possess an accompanying AP-lyase activity that cleaves the sugar-phosphate chain of DNA. Since the first discovery of a DNA glycosylase, many studies have elucidated the mechanisms of action, substrate specificities and excision kinetics of these enzymes present in all living organisms. For this purpose, most studies used single- or double-stranded oligodeoxynucleotides with a single DNA lesion embedded at a defined position. High-molecular weight DNA with multiple base lesions has been used in other studies with the advantage of the simultaneous investigation of many DNA base lesions as substrates. Differences between the substrate specificities and excision kinetics of DNA glycosylases have been found when these two different substrates were used. Some DNA glycosylases possess varying substrate specificities for either purine-derived lesions or pyrimidine-derived lesions, whereas others exhibit cross-activity for both types of lesions. Laboratory animals with knockouts of the genes of DNA glycosylases have also been used to provide unequivocal evidence for the substrates, which had previously been found in in vitro studies, to be the actual substrates in vivo as well. On the basis of the knowledge gained from the past studies, efforts are being made to discover small molecule inhibitors of DNA glycosylases that may be used as potential drugs in cancer therapy., (Published by Elsevier B.V.)

Oxidatively induced DNA damage is caused in living organisms by a variety of damaging agents, resulting in the formation of a multiplicity of lesions, which are mutagenic and cytotoxic. Unless repaired by DNA repair mechanisms before DNA replication, DNA lesions can lead to genomic instability, which is one of the hallmarks of cancer. Oxidatively induced DNA damage is mainly repaired by base excision repair pathway with the involvement of a plethora of proteins. Cancer tissues develop greater DNA repair capacity than normal tissues by overexpressing DNA repair proteins. Increased DNA repair in tumors that removes DNA lesions generated by therapeutic agents before they became toxic is a major mechanism in the development of therapy resistance. Evidence suggests that DNA repair capacity may be a predictive biomarker of patient response. Thus, knowledge of DNA-protein expressions in disease-free and cancerous tissues may help predict and guide development of treatments and yield the best therapeutic response. Our laboratory has developed methodologies that use mass spectrometry with isotope dilution for the measurement of expression of DNA repair proteins in human tissues and cultured cells. For this purpose, full-length (15)N-labeled analogs of a number of human DNA repair proteins have been produced and purified to be used as internal standards for positive identification and accurate quantification. This chapter describes in detail the protocols of this work. The use of (15)N-labeled proteins as internal standards for the measurement of several DNA repair proteins in vivo is also presented., (© 2016 Elsevier Inc. All rights reserved.)

The comet assay is widely used in human biomonitoring to measure DNA damage as a marker of exposure to genotoxic agents or to investigate genoprotective effects. Studies often involve small numbers of subjects, and design may be sub-optimal in other respects. In addition, comet assay protocols in use in different laboratories vary significantly. In spite of these difficulties, it is appropriate to carry out a pooled analysis of all available comet assay biomonitoring data, in order to establish baseline parameters of DNA damage, and to investigate associations between comet assay measurements and factors such as sex, age, smoking status, nutrition, lifestyle, etc. With this as its major objective, the ComNet project has recruited almost 100 research groups willing to share datasets. Here we provide a background to this project, discussing the history of the comet assay and practical issues that can critically affect its performance. We survey its diverse applications in biomonitoring studies, including environmental and occupational exposure to genotoxic agents, genoprotection by dietary and other factors, DNA damage associated with various diseases, and intrinsic factors that affect DNA damage levels in humans. We examine in depth the quality of data from a random selection of studies, from an epidemiological and statistical point of view., (Copyright © 2013 Elsevier B.V. All rights reserved.)