Determination of N7-glycidamide guanine adducts in human blood DNA following exposure to dietary acrylamide using liquid chromatography/tandem mass spectrometry.

RATIONALE: Acrylamide is classified as a probable human carcinogen that is metabolised to glycidamide, which can covalently bind to DNA. The aim of this study was to investigate the formation of N7-glycidamide guanine (N7-GA-Gua) adducts in human blood DNA following exposure to acrylamide present in carbohydrate-rich foods as part of the normal human diet. METHODS: Lymphocyte DNA was extracted from blood samples obtained from healthy human volunteers. Following thermal depurination of the DNA samples, N7-GA-Gua adducts were quantified using a validated liquid chromatography/tandem mass spectrometry (LC/MS/MS) method incorporating a stable isotope labelled internal standard. Estimated dietary acrylamide intake was recorded by completion of food frequency questionnaires for the 24 hours prior to volunteer blood donation. RESULTS: An LC/MS/MS method was validated with a limit of detection of 0.25 fmol and a lower limit of quantitation of 0.50 fmol on column. N7-GA-Gua adducts were detected in human blood DNA with the levels ranging between 0.3 to 6.3 adducts per 108 nucleotides. The acrylamide intake was calculated from the food frequency questionnaires ranging between 20.0 and 78.6 µg. CONCLUSIONS: Identification and quantification of N7-GA-Gua adducts in the blood DNA of healthy volunteers suggests that dietary acrylamide exposure may lead to the formation of DNA adducts. This important finding warrants further investigation to ascertain a correlation between environmental/dietary acrylamide exposure and levels of DNA adducts.

Maternal diet during pregnancy and micronuclei frequency in peripheral blood T lymphocytes in mothers and newborns (Rhea cohort, Crete).

PURPOSE: The study assessed whether diet and adherence to cancer prevention guidelines during pregnancy were associated with micronucleus (MN) frequency in mothers and newborns. MN is biomarkers of early genetic effects that have been associated with cancer risk in adults. METHODS: A total of 188 mothers and 200 newborns from the Rhea cohort (Greece) were included in the study. At early-mid pregnancy, we conducted personal interviews and a validated food frequency questionnaire was completed. With this information, we constructed a score reflecting adherence to the World Cancer Research Fund/American Institute for Cancer Research cancer prevention guidelines on diet, physical activity and body fatness. At delivery, maternal and/or cord blood was collected to measure DNA and hemoglobin adducts of dietary origin and frequencies of MN in binucleated and mononucleated T lymphocytes (MNBN and MNMONO). RESULTS: In mothers, higher levels of red meat consumption were associated with increased MNBN frequency [2nd tertile IRR = 1.34 (1.00, 1.80), 3rd tertile IRR = 1.33 (0.96, 1.85)] and MNMONO frequency [2nd tertile IRR = 1.53 (0.84, 2.77), 3rd tertile IRR = 2.69 (1.44, 5.05)]. The opposite trend was observed for MNBN in newborns [2nd tertile IRR = 0.64 (0.44, 0.94), 3rd tertile IRR = 0.68 (0.46, 1.01)], and no association was observed with MNMONO. Increased MN frequency in pregnant women with high red meat consumption is consistent with previous knowledge. CONCLUSIONS: Our results also suggest exposure to genotoxics during pregnancy might affect differently mothers and newborns. The predictive value of MN as biomarker for childhood cancer, rather than adulthood, remains unclear. With few exceptions, the association between maternal carcinogenic exposures during pregnancy and childhood cancer or early biologic effect biomarkers remains poorly understood.

Antitumour benzothiazoles. Part 32: DNA adducts and double strand breaks correlate with activity; synthesis of 5F203 hydrogels for local delivery.

Potent, selective antitumour AhR ligands 5F 203 and GW 610 are bioactivated by CYPs 1A1 and 2W1. Herein we reason that DNA adducts' generation resulting in lethal DNA double strand breaks (DSBs) underlies benzothiazoles' activity. Treatment of sensitive carcinoma cell lines with GW 610 generated co-eluting DNA adducts (R(2)>0.7). Time-dependent appearance of γ-H2AX foci revealed subsequent DNA double strand breaks. Propensity for systemic toxicity of benzothiazoles steered development of prodrugs' hydrogels for localised delivery. Clinical applications of targeted therapies include prevention or treatment of recurrent disease after surgical resection of solid tumours. In vitro evaluation of 5F 203 prodrugs' activity demonstrated nanomolar potency against MCF-7 breast and IGROV-1 ovarian carcinoma cell lines.

An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride.

The framework analysis previously presented for using DNA adduct information in the risk assessment of chemical carcinogens was applied in a series of case studies which place the adduct information into context with the key events in carcinogenesis to determine whether they could be used to support a mutagenic mode of action (MOA) for the examined chemicals. Three data-rich chemicals, aflatoxin B1 (AFB1), tamoxifen (Tam) and vinyl chloride (VCl) were selected for this exercise. These chemicals were selected because they are known human carcinogens and have different characteristics: AFB1 forms a unique adduct and human exposure is through contaminated foods; Tam is a pharmaceutical given to women so that the dose and duration of exposure are known, forms unique adducts in rodents, and has both estrogenic and genotoxic properties; and VCl, to which there is industrial exposure, forms a number of adducts that are identical to endogenous adducts found in unexposed people. All three chemicals produce liver tumors in rats. AFB1 and VCl also produce liver tumors in humans, but Tam induces human uterine tumors, only. To support a mutagenic MOA, the chemical-induced adducts must be characterized, shown to be pro-mutagenic, be present in the tumor target tissue, and produce mutations of the class found in the tumor. The adducts formed by AFB1 and VCl support a mutagenic MOA for their carcinogenicity. However, the data available for Tam shows a mutagenic MOA for liver tumors in rats, but its carcinogenicity in humans is most likely via a different MOA.

STrengthening the Reporting of OBservational studies in Epidemiology - Molecular Epidemiology (STROBE-ME): an extension of the STROBE statement.

Advances in laboratory techniques have led to a rapidly increasing use of biomarkers in epidemiological studies. Biomarkers of internal dose, early biological change, susceptibility and clinical outcomes are used as proxies for investigating interactions between external and/or endogenous agents and body components or processes. The need for improved reporting of scientific research led to influential statements of recommendations such as the STrengthening Reporting of OBservational studies in Epidemiology (STROBE) statement. The STROBE initiative established in 2004 aimed to provide guidance on how to report observational research. Its guidelines provide a user-friendly checklist of 22 items to be reported in epidemiological studies, with items specific to the three main study designs: cohort studies, case-control studies and cross-sectional studies. The present STrengthening the Reporting of OBservational studies in Epidemiology -Molecular Epidemiology (STROBE-ME) initiative builds on the STROBE statement implementing nine existing items of STROBE and providing 17 additional items to the 22 items of STROBE checklist. The additions relate to the use of biomarkers in epidemiological studies, concerning collection, handling and storage of biological samples; laboratory methods, validity and reliability of biomarkers; specificities of study design; and ethical considerations. The STROBE-ME recommendations are intended to complement the STROBE recommendations.

STrengthening the Reporting of OBservational studies in Epidemiology--Molecular Epidemiology (STROBE-ME): an extension of the STROBE statement.

Advances in laboratory techniques have led to a rapidly increasing use of biomarkers in epidemiological studies. Biomarkers of internal dose, early biological change, susceptibility and clinical outcomes are used as proxies for investigating interactions between external and / or endogenous agents and body components or processes. The need for improved reporting of scientific research led to influential statements of recommendations such as the STrengthening Reporting of OBservational studies in Epidemiology (STROBE) statement. The STROBE initiative established in 2004 aimed to provide guidance on how to report observational research. Its guidelines provide a user-friendly checklist of 22 items to be reported in epidemiological studies, with items specific to the three main study designs: cohort studies, case-control studies and cross-sectional studies. The present STrengthening the Reporting of OBservational studies in Epidemiology - Molecular Epidemiology (STROBE-ME) initiative builds on the STROBE statement implementing nine existing items of STROBE and providing 17 additional items to the 22 items of STROBE checklist. The additions relate to the use of biomarkers in epidemiological studies, concerning collection, handling and storage of biological samples; laboratory methods, validity and reliability of biomarkers; specificities of study design; and ethical considerations. The STROBE-ME recommendations are intended to complement the STROBE recommendations.

Detection of acetaldehyde derived N(2)-ethyl-2'-deoxyguanosine in human leukocyte DNA following alcohol consumption.

Epidemiological studies have shown an association between alcohol (ethanol) consumption and increased cancer risk. The effect of alcohol consumption on the levels and persistence of N(2)-ethylidene-2'-deoxyguanosine (N(2)-ethylidene-dG) formed by acetaldehyde, the oxidative metabolite of ethanol, in human leukocyte DNA was investigated. DNA was isolated from venous blood samples obtained from 30 male non-smoking individuals before consumption of alcohol (0h) and subsequently at 3-5h following the consumption of 150mL of vodka (containing 42% pure ethanol). Additional samples were collected 24h and 48h post-alcohol consumption. The levels of N(2)-ethyl-2'-deoxyguanosine (N(2)-ethyl-dG) in the DNA were determined following reduction of N(2)-ethylidene-dG with sodium cyanoborohydride using a liquid chromatography-tandem mass spectrometry selected reaction monitoring method. A slight time-dependent trend showing an increase and decrease in the levels of N(2)-ethyl-dG was observed following consumption of alcohol compared to time 0h, however, the differences were not statistically significant. The average levels of N(2)-ethyl-dG observed at 0h, 3-5h, 24h and 48h time points following ingestion of alcohol were 34.6±21.9, 35.1±21.0, 36.8±20.7 and 35.6±21.1 per 10(8) 2'-deoxynucleosides, respectively. In conclusion, alcohol consumption that could be encountered under social drinking conditions, does not significantly alter the levels of the acetaldehyde derived DNA adduct, N(2)-ethyl-dG in human leukocyte DNA from healthy individuals.

Effect of hepatic cytochrome P450 (P450) oxidoreductase deficiency on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-DNA adduct formation in P450 reductase conditional null mice.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), formed during the cooking of foods, induces colon cancer in rodents. PhIP is metabolically activated by cytochromes P450 (P450s). To evaluate the role of hepatic P450s in the bioactivation of PhIP, we used Reductase Conditional Null (RCN) mice, in which cytochrome P450 oxidoreductase (POR), the unique electron donor to P450s, can be specifically deleted in hepatocytes by pretreatment with 3-methylcholanthrene (3-MC), resulting in the loss of essentially all hepatic P450 function. RCN mice were treated orally with 50 mg/kg b.wt. PhIP daily for 5 days, with and without 3-MC pretreatment. PhIP-DNA adducts (i.e., N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine [dG-C8-PhIP]), measured by liquid chromatography-tandem mass spectrometry, were highest in colon (1362 adducts/10(8) deoxynucleosides), whereas adduct levels in liver were ∼3.5-fold lower. Whereas no differences in PhIP-DNA adduct levels were found in livers with active POR versus inactivated POR, adduct levels were on average ∼2-fold lower in extrahepatic tissues of mice lacking hepatic POR. Hepatic microsomes from RCN mice with or without 3-MC pretreatment were also incubated with PhIP and DNA in vitro. PhIP-DNA adduct formation was ∼8-fold lower with hepatic microsomes from POR-inactivated mice than with those with active POR. Most of the hepatic microsomal activation of PhIP in vitro was attributable to CYP1A. Our results show that PhIP-DNA adduct formation in colon involves hepatic N-oxidation, circulation of activated metabolites via the bloodstream to extrahepatic tissues, and further activation, resulting in the formation of dG-C8-PhIP. Besides hepatic P450s, PhIP may be metabolically activated mainly by a non-P450 pathway in liver.

STrengthening the Reporting of OBservational studies in Epidemiology - Molecular Epidemiology (STROBE-ME): an extension of the STROBE statement.

Advances in laboratory techniques have led to a rapidly increasing use of biomarkers in epidemiological studies. Biomarkers of internal dose, early biological change, susceptibility and clinical outcomes are used as proxies for investigating the interactions between external and/or endogenous agents and the body components or processes. The need for improved reporting of scientific research led to influential statements of recommendations such as the STrenghtening Reporting of Observational studies in Epidemiology (STROBE) statement. The STROBE initiative established in 2004 aimed to provide guidance on how to report observational research. Its guidelines provide a user-friendly checklist of 22 items to be reported in epidemiological studies, with items specific to the three main study designs: cohort studies, case-control studies and cross-sectional studies. The present STrengthening the Reporting of OBservational studies in Epidemiology - Molecular Epidemiology (STROBE-ME) initiative builds on the STROBE Statement implementing 9 existing items of STROBE and providing 17 additional items to the 22 items of STROBE checklist. The additions relate to the use of biomarkers in epidemiological studies, concerning collection, handling and storage of biological samples; laboratory methods, validity and reliability of biomarkers; specificities of study design; and ethical considerations. The STROBE-ME recommendations are intended to complement the STROBE recommendations.

Evaluation of urinary ribonucleoside profiling for clinical biomarker discovery using constant neutral loss scanning liquid chromatography/tandem mass spectrometry.

The patterns and levels of urinary excreted ribonucleosides which reflect RNA turnover and metabolism in humans offer the potential for early detection of disease and monitoring of therapeutic intervention. A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method employing constant neutral loss (CNL) scanning for the loss of the ribose moiety (132 u) was used to detect ribonucleosides in human urine and to evaluate this analytical platform for biomarker research in clinical trials. Ribonucleosides were stable and not influenced by the time spent at room temperature prior to freezing or long-term storage at -80 °C. Matrix effects caused variation in the mass spectrometer response which was dependent on the concentration of the analysed urine sample. For the use of urinary ribonucleoside profiling in clinical biomarker studies, adjustment of the urine samples to a common concentration prior to sample preparation is therefore advocated. Changes in the mass spectrometer response should be accounted for by the use of an internal standard added after sample preparation. Diurnal variation exceeded inter-day variation of an individual's ribonucleoside profile, but inter-person differences were predominant and allowed the separation of individuals against each other in a multivariate space. Due to considerable diurnal variation the use of spot urine samples would introduce unnecessary variation and should be replaced by the collection of multiple spot urine samples across the day, where possible. Should such a protocol not be feasible, biological intra-day and inter-day variation must be considered and accounted for in the data interpretation.

STrengthening the reporting of OBservational studies in Epidemiology-Molecular Epidemiology (STROBE-ME): an extension of the STROBE statement.

Advances in laboratory techniques have led to a rapidly increasing use of biomarkers in epidemiological studies. Biomarkers of internal dose, early biological change, susceptibility, and clinical outcomes are used as proxies for investigating the interactions between external and/or endogenous agents and the body components or processes. The need for improved reporting of scientific research led to influential statements of recommendations such as STrengthening Reporting of Observational studies in Epidemiology (STROBE) statement. The STROBE initiative established in 2004 aimed to provide guidance on how to report observational research. Its guidelines provide a user-friendly checklist of 22 items to be reported in epidemiological studies, with items specific to the three main study designs: cohort studies, case-control studies and cross-sectional studies. The present STrengthening the Reporting of OBservational studies in Epidemiology-Molecular Epidemiology (STROBE-ME) initiative builds on the STROBE Statement implementing 9 existing items of STROBE and providing 17 additional items to the 22 items of STROBE checklist. The additions relate to the use of biomarkers in epidemiological studies, concerning collection, handling and storage of biological samples; laboratory methods, validity and reliability of biomarkers; specificities of study design; and ethical considerations. The STROBE-ME recommendations are intended to complement the STROBE recommendations.

Strategies in case of positive in vivo results in genotoxicity testing.

At the 2009 International Workshop on Genotoxicity Testing in Basel, an expert group gathered to provide guidance on suitable follow-up tests to describe risk when basic in vivo genotoxicity tests have yielded positive results. The working group agreed that non-linear dose-response curves occur in vivo with at least some DNA-reactive agents. Quantitative risk assessment in such cases requires the use of (1) adequate data, i.e., the use of all available data for the selection of reliable in vivo models to be used for quantitative risk assessment, (2) appropriate mathematical models and statistical analysis for characterizing the dose-response relationships and allowing the use of quantitative and dose-response information in the interpretation of results, (3) mode of action (MOA) information for the evaluation and analysis of risk, and (4) reliable assessments of the internal dose across species for deriving acceptable margins of exposure and risk levels. Hence, the elucidation of MOA and understanding of the mechanism underlying the dose-response curve are important components of risk assessment. The group agreed on the need for (i) the development of in vivo assays, especially multi-endpoint, multi-species assays, with emphasis on those applicable to humans, and (ii) consensus about the most appropriate mathematical models and statistical analyses for defining non-linear dose-responses and exposure levels associated with acceptable risk.

Determination of cisplatin 1,2-intrastrand guanine-guanine DNA adducts in human leukocytes by high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry.

Platinum-containing drugs are widely used to treat cancer in a variety of clinical settings. Their mode of action involves the formation of DNA adducts, which facilitate apoptosis in cancer cells. Cisplatin binds to the N7 position of the purine DNA bases forming intrastrand cross-links between either two adjacent guanines [cis-Pt(NH(3))(2)d(pGpG), 1,2-GG] or an adjacent adenine and guanine [cis-Pt(NH(3))(2)d(pApG), 1,2-AG)]. The cytotoxic efficacy for each of the different types of DNA adducts and the relationship between adduct levels in tumor cells and blood are not well understood. By using these Pt-containing adduct species as biomarkers, information on a patient's response to chemotherapy would be directly related to the mode of action of the drug. This type of analysis requires the most sensitive and specific methods available, to facilitate detection limits sufficient to measure the DNA adduct in the limited sample quantities available from patients. This was achieved in the current study by coupling a highly specific enzyme-based adduct isolation method with a sensitive detection system based on HPLC coupled to inductively coupled plasma mass spectrometry to measure the 1,2-GG cisplatin adducts formed in DNA. The method was developed and validated using calf thymus DNA and two different adenocarcinoma cell lines. The values for the limit of detection (LOD) and the limit of quantitation determined for the 1,2-GG cisplatin adduct were 0.21 and 0.67 fmol per microg DNA, respectively. This corresponds to an absolute LOD of 0.8 pg as Pt for the 1,2-GG adduct. Cisplatin-sensitive (H23) and -resistant (A549) tumor cells were exposed to the drug, and the 1,2-GG adduct levels were measured over a 24 h time period. The results showed a statistically significant (P < 0.05) higher concentration in the sensitive cells as compared to the resistant cells after repair for 7 h. Although the adduct concentration present fell at subsequent time points (12 and 24 h), the levels in each cell line were broadly similar. The protocol was then applied to the analysis of patient samples taken before and then 1 h after treatment. The 1,2-GG cisplatin adduct was present in the range from 113 to 1245 fg Pt per microg DNA in all of the patient samples taken after treatment. Although the adduct was not present at levels greater than the LOD in the initial pretreatment samples, trace amounts were discernible in some patient samples on their third treatment cycle.

Phosphotriester adducts (PTEs): DNA's overlooked lesion.

In addition to reacting with DNA base moieties, many chemical genotoxins also react with the oxygen atoms of the internucleotidic phosphodiester linkages to form phosphotriester adducts (PTEs). In view of their stability under physiological conditions, it has been suggested that PTEs may be useful biomarkers for measuring cumulative genotoxin exposure. The methodology for their determination is varied and still not completely developed but includes determination of hydrolysis products and (32)P-postlabelling approaches. More recently, transalkylation and direct mass spectrometry techniques have been devised, which give extra chemical information on the structures of the PTEs. The proportion of DNA damage formed as PTEs is much greater with SN1 compared to SN2 alkylating agents, and it has been shown in DNA that the formation of PTEs is partially sequence dependent. PTEs have been considered to be refractory to repair in mammalian cells but repair mechanisms have been found in prokaryotic cells, e.g. PTEs in Escherichia coli are repaired by O(6)-methylguanine-DNA methyltransferase (O(6)-MGT or Ada protein). However, studies on in vivo persistence of PTEs in mammalian systems have not ruled out the possibility of a contribution from an active repair process for PTEs. The biological significance of PTEs is largely unstudied and unknown, although effects of PTEs on DNA polymerases, and some exo- and endonucleases have been observed. Also site-specific PTEs impair the repair processing of adjacent sites of DNA damage, which may be a biological mechanism of importance for these lesions. In this review, we will consider the analytical methods available for the determination of PTEs, their stability in vitro and in vivo, the mechanisms for their repair, their possible biological significance and their potential role as biomarkers in human molecular epidemiology studies.

Development of a targeted adductomic method for the determination of polycyclic aromatic hydrocarbon DNA adducts using online column-switching liquid chromatography/tandem mass spectrometry.

Human exposure to polycyclic aromatic hydrocarbons (PAHs) from sources such as industrial or urban air pollution, tobacco smoke and cooked food is not confined to a single compound, but instead to mixtures of different PAHs. The interaction of different PAHs may lead to additive, synergistic or antagonistic effects in terms of DNA adduct formation and carcinogenic activity resulting from changes in metabolic activation to reactive intermediates and DNA repair. The development of a targeted DNA adductomic approach using liquid chromatography/tandem mass spectrometry (LC/MS/MS) incorporating software-based peak picking and integration for the assessment of exposure to mixtures of PAHs is described. For method development PAH-modified DNA samples were obtained by reaction of the anti-dihydrodiol epoxide metabolites of benzo[a]pyrene, benzo[b]fluoranthene, dibenzo[a,l]pyrene (DB[a,l]P) and dibenz[a,h]anthracene with calf thymus DNA in vitro and enzymatically hydrolysed to 2'-deoxynucleosides. Positive LC/electrospray ionisation (ESI)-MS/MS collision-induced dissociation product ion spectra data showed that the majority of adducts displayed a common fragmentation for the neutral loss of 116 u (2'-deoxyribose) resulting in a major product ion derived from the adducted base. The exception was the DB[a,l]P dihydrodiol epoxide adduct of 2'-deoxyadenosine which resulted in major product ions derived from the PAH moiety being detected. Specific detection of mixtures of PAH-adducted 2'-deoxynucleosides was achieved using online column-switching LC/MS/MS in conjunction with selected reaction monitoring (SRM) of the [M+H](+) to [M+H-116](+) transition plus product ions derived from the PAH moiety for improved sensitivity of detection and a comparison was made to detection by constant neutral loss scanning. In conclusion, different PAH DNA adducts were detected by employing SRM [M+H-116](+) transitions or constant neutral loss scanning. However, for improved sensitivity of detection optimised SRM transitions relating to the PAH moiety product ions are required for certain PAH DNA adducts for the development of targeted DNA adductomic methods.

Low 8-oxo-7,8-dihydro-2'-deoxyguanosine levels and influence of genetic background in an Andean population exposed to high levels of arsenic.

BACKGROUND: Arsenic (As) causes oxidative stress through generation of reactive oxygen species. 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a sensitive marker of oxidative DNA damage, has been associated with As exposure in some studies, but not in others, possibly due to population-specific genetic factors. OBJECTIVES: To evaluate the association between As and 8-oxodG in urine in a population with a low urinary monomethylated As (%MMA) and high dimethylated As (%DMA), as well as the genetic impact on (a) 8-oxodG concentrations and (b) the association between As and 8-oxodG. MATERIALS AND METHODS: Women (N=108) in the Argentinean Andes were interviewed and urine was analyzed for arsenic metabolites (ICPMS) and 8-oxodG (LC-MS/MS). Twenty-seven polymorphisms in genes related to oxidative stress and one in As(+III)methyltransferase (AS3MT) were studied. RESULTS: Median concentration of 8-oxodG was 4.7 nmol/L (adjusted for specific weight; range 1.6-13, corresponding to 1.7 microg/g creatinine, range 0.57-4.8) and of total urinary As metabolites (U-As) 290 microg/L (range 94-720; 380 microg/g creatinine, range 140-1100). Concentrations of 8-oxodG were positively associated with %MMA (strongest association, p=0.013), and weakly associated with U-As (positively) and %DMA (negatively). These associations were strengthened when taking ethnicity into account, possibly reflecting genetic differences in As metabolism and genes regulating oxidative stress and DNA maintenance. A genetic influence on 8-oxodG concentrations was seen for polymorphisms in apurinic/apyrimidinic endonuclease 1 (APEX1), DNA-methyltransferases 1 and 3b (DNMT1, DNMT3B), thioredoxin reductase 1 (TXNRD1) and 2 (TXNRD2) and glutaredoxin (GLRX). CONCLUSION: Despite high As exposure, the concentrations of 8-oxodG in this population were low compared with other As-exposed populations studied. The strongest association was found for %MMA, stressing that some inconsistencies between As and 8-oxodG partly depend on population variations in As metabolism. We found evidence of genetic impact on 8-oxodG concentrations.

Mutagenicity of tamoxifen DNA adducts in human endometrial cells and in silico prediction of p53 mutation hotspots.

Tamoxifen elevates the risk of endometrial tumours in women and alpha-(N(2)-deoxyguanosinyl)-tamoxifen adducts are reportedly present in endometrial tissue of patients undergoing therapy. Given the widespread use of tamoxifen there is considerable interest in elucidating the mechanisms underlying treatment-associated cancer. Using a combined experimental and multivariate statistical approach we have examined the mutagenicity and potential consequences of adduct formation by reactive intermediates in target uterine cells. pSP189 plasmid containing the supF gene was incubated with alpha-acetoxytamoxifen or 4-hydroxytamoxifen quinone methide (4-OHtamQM) to generate dG-N(2)-tamoxifen and dG-N(2)-4-hydroxytamoxifen, respectively. Plasmids were replicated in Ishikawa cells then screened in Escherichia coli. Treatment with both alpha-acetoxytamoxifen and 4-OHtamQM caused a dose-related increase in adduct levels, resulting in a damage-dependent increase in mutation frequency for alpha-acetoxytamoxifen; 4-OHtamQM had no apparent effect. Only alpha-acetoxytamoxifen generated statistically different supF mutation spectra relative to the spontaneous pattern, with most mutations being GC-->TA transversions. Application of the LwPy53 algorithm to the alpha-acetoxytamoxifen spectrum predicted strong GC-->TA hotspots at codons 244 and 273. These signature alterations do not correlate with current reports of the mutations observed in endometrial carcinomas from treated women, suggesting that dG-N(2)-tam adduct formation in the p53 gene is not a prerequisite for endometrial cancer initiation in women.

Evaluation of the DNA damaging potential of cannabis cigarette smoke by the determination of acetaldehyde derived N2-ethyl-2'-deoxyguanosine adducts.

Acetaldehyde is an ubiquitous genotoxic compound that has been classified as a possible carcinogen to humans. It can react with DNA to form primarily a Schiff base N(2)-ethylidene-2'-deoxyguanosine (N(2)-ethylidene-dG) adduct. An online column-switching valve liquid chromatography tandem mass spectrometry (LC-MS/MS) selected reaction monitoring (SRM) method was developed for the determination of N(2)-ethylidene-dG adducts in DNA following reduction with sodium cyanoborohydride (NaBH(3)CN) to the chemically stable N(2)-ethyl-2'-deoxyguanosine (N(2)-ethyl-dG) adduct. Accurate quantitation of the adduct was obtained by the addition of the [(15)N(5)]N(2)-ethyl-dG stable isotope-labeled internal standard prior to enzymatic hydrolysis of the DNA samples to 2'-deoxynucleosides with the incorporation of NaBH(3)CN in the DNA hydrolysis buffer. The method required 50 microg of hydrolyzed DNA on column for the analysis, and the limit of detection for N(2)-ethyl-dG was 2.0 fmol. The analysis of calf thymus DNA treated in vitro with acetaldehyde (ranging from 0.5 to 100 mM) or with the smoke generated from 1, 5, and 10 cannabis cigarettes showed linear dose-dependent increases in the level of N(2)-ethyl-dG adducts (r = 0.954 and r = 0.999, respectively). Similar levels (332.8 +/- 21.9 vs 348.4 +/- 19.1 adducts per 10(8) 2'-deoxynucleosides) of N(2)-ethyl-dG adducts were detected following the exposure of calf thymus DNA to 10 tobacco or 10 cannabis cigarettes. No significant difference was found in the levels of N(2)-ethyl-dG adducts in human lung DNA obtained from nonsmokers (n = 4) and smokers (n = 4) with the average level observed as 13.3 +/- 0.7 adducts per 10(8) 2'-deoxynucleosides. No N(2)-ethyl-dG adducts were detected in any of the DNA samples following analysis with the omission of NaBH(3)CN from the DNA hydrolysis buffer. In conclusion, these results provide evidence for the DNA damaging potential of cannabis smoke, implying that the consumption of cannabis cigarettes may be detrimental to human health with the possibility to initiate cancer development.

Quantification of 3-nitrobenzanthrone-DNA adducts using online column-switching HPLC-electrospray tandem mass spectrometry.

The aromatic nitroketone 3-nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one; 3-NBA) is an extremely potent mutagen and a suspected human carcinogen detected in the exhaust of diesel engines and in airborne particulate matter. 3-NBA is metabolically activated via reduction of the nitro group to the hydroxylamine (N-OH-3-ABA) to form covalent DNA adducts. Thus far, the detection and quantification of covalent 3-NBA-DNA adducts has relied solely on (32)P-postlabeling methodologies. In order to expand the range of available techniques for the detection and improved quantification of 3-NBA-DNA adducts, we have developed a method based upon online column-switching HPLC coupled to electrospray tandem mass spectrometry, with isotopic dilution of (15)N-labeled internal standards. This methodology was applied to the determination of three 3-NBA-derived adducts: 2-(2'-deoxyguanosin-N(2)-yl)-3-aminobenzanthrone (dG-N(2)-3-ABA), N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-N-3-ABA) and 2-(2'-deoxyguanosine-8-yl)-3-aminobenzanthrone (dG-C8-C2-3-ABA). Dose-dependent increases were observed for all three adducts when salmon testis DNA was reacted with N-acetoxy-3-aminobenzanthrone (N-AcO-3-ABA). dG-C8-C2-3-ABA was detected at much lower levels (overall 1%) than the other two adducts. DNA samples isolated from tissues of rats treated either intratracheally with 3-NBA or intraperitoneally with N-OH-3-ABA were analyzed by mass spectrometry, and the results compared to those obtained by (32)P-postlabeling. The method required 50 microg of hydrolyzed animal DNA on column and the limit of detection was 2.0 fmol for each adduct. dG-C8-C2-3-ABA was not observed in any of the samples providing confirmation that it is not formed in vivo. Linear regression analysis of the levels of dG-N(2)-3-ABA and dG-C8-N-3-ABA in the rat DNA showed a reasonable correlation between the two methods (R(2) = 0.88 and 0.93, respectively). In summary, the mass spectrometric method is a faster, more automated analytical approach that also provides structural confirmation of the adducts detected by (32)P-postlabeling, and it has sufficient sensitivity and precision to analyze DNA adducts in animals exposed to 3-NBA or its hydroxylamine metabolite.

Creating context for the use of DNA adduct data in cancer risk assessment: II. Overview of methods of identification and quantitation of DNA damage.

The formation of deoxyribonucleic acid (DNA) adducts can have important and adverse consequences for cellular and whole organism function. Available methods for identification of DNA damage and quantification of adducts are reviewed. Analyses can be performed on various samples including tissues, isolated cells, and intact or hydrolyzed (digested) DNA from a variety of biological samples of interest for monitoring in humans. Sensitivity and specificity are considered key factors for selecting the type of method for assessing DNA perturbation. The amount of DNA needed for analysis is dependent upon the method and ranges widely, from <1 microg to 3 mg. The methods discussed include the Comet assay, the ligation-mediated polymerase reaction, histochemical and immunologic methods, radiolabeled ((14)C- and (3)H-) binding, (32)P-postlabeling, and methods dependent on gas chromatography (GC) or high-performance liquid chromatography (HPLC) with detection by electron capture, electrochemical detection, single or tandem mass spectrometry, or accelerator mass spectrometry. Sensitivity is ranked, and ranges from approximately 1 adduct in 10(4) to 10(12) nucleotides. A brief overview of oxidatively generated DNA damage is also presented. Assay limitations are discussed along with issues that may have impact on the reliability of results, such as sample collection, processing, and storage. Although certain methodologies are mature, improving technology will continue to enhance the specificity and sensitivity of adduct analysis. Because limited guidance and recommendations exist for adduct analysis, this effort supports the HESI Committee goal of developing a framework for use of DNA adduct data in risk assessment.