• Detection and quantitation of N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine adducts in DNA using online column-switching liquid chromatography tandem mass spectrometry.

      Singh, Rajinder; Arlt, Volker M.; Henderson, Colin J.; Phillips, David H.; Farmer, Peter B.; Gamboa da Costa, Goncalo (2010-08-01)
      The heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed by the grilled cooking of certain foods such as meats, poultry and fish. PhIP has been shown to induce tumours in the colon, prostate and mammary glands of rats and is regarded as a potential human dietary carcinogen. PhIP is metabolically activated via cytochrome P450 mediated oxidation to an N-hydroxylamino-PhIP intermediate that is subsequently converted to an ester by N-acetyltransferases or sulfotransferases and undergoes heterolytic cleavage to produce a PhIP-nitrenium ion, which reacts with DNA to form the N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP-C8-dG) adduct. Thus far, the detection and quantification of PhIP-DNA adducts has relied to a large extent on (32)P-postlabelling methodologies. In order to expand the array of available techniques for the detection and improved quantification of PhIP-C8-dG adducts in DNA we have developed an online column-switching liquid chromatography (LC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) selected reaction monitoring (SRM) method incorporating an isotopically [(13)C(10)]-labelled PhIP-C8-dG internal standard for the analysis of DNA enzymatically hydrolysed to 2'-deoxynucleosides. A dose-dependent increase was observed for PhIP-C8-dG adducts when salmon testis DNA was reacted with N-acetoxy-PhIP. Analysis of DNA samples isolated from colon tissue of mice treated by oral gavage daily for 5 days with 50 mg/kg body weight of PhIP resulted in the detection of an average level of 14.8+/-3.7 PhIP-C8-dG adducts per 10(6) 2'-deoxynucleosides. The method required 50 microg of hydrolysed animal DNA on column and the limit of detection for PhIP-C8-dG was 2.5 fmol (1.5 PhIP-C8-dG adducts per 10(8) 2'-deoxynucleosides). In summary, the LC-ESI-MS/MS SRM method provides for the rapid automation of the sample clean up and a reduction in matrix components that would otherwise interfere with the mass spectrometric analysis, with sufficient sensitivity and precision to analyse DNA adducts in animals exposed to PhIP.
    • Detection of acetaldehyde derived N(2)-ethyl-2'-deoxyguanosine in human leukocyte DNA following alcohol consumption.

      Singh, Rajinder; Gromadzinska, Jolanta; Mistry, Yogita; Cordell, Rebecca; Juren, Tina; Segerback, Dan; Farmer, Peter B. (2012-09-01)
      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.
    • Determination of 8-oxo-2'-deoxyguanosine and creatinine in murine and human urine by liquid chromatography/tandem mass spectrometry: application to chemoprevention studies.

      Teichert, Friederike; Verschoyle, Richard D.; Greaves, Peter; Thorpe, James F.; Mellon, J. Kilian; Steward, William P.; Farmer, Peter B.; Gescher, Andreas J.; Singh, Rajinder (2009-01)
      Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) represents a non-invasive biomarker for oxidative stress and may be useful for monitoring chemotherapeutic and chemopreventive interventions associated with cancer-related alterations in oxidative stress. We describe the development and validation of two separate liquid chromatography/tandem mass spectrometry (LC/MS/MS) selected reaction monitoring (SRM) methods for the determination of 8-oxodG and creatinine in both murine and human urine using stable isotope labelled internal standards. Levels of 8-oxodG were normalised to creatinine. The LC/MS/MS methods were applied to two chemoprevention studies utilising tea polyphenols in humans and TRAMP (TRansgenic Adenocarcinoma of the Mouse Prostate) mice. Patients with benign prostatic hyperplasia received 1 g/day of green tea polyphenols (GTP), 1 g/day of black tea theaflavins (BTT) or no treatment for 4 weeks. TRAMP mice received GTP (0.05% in drinking water) for 4 or 25 weeks. Prostate pathology in TRAMP mice was not affected by GTP. Levels of 8-oxodG were not altered by tea polyphenols in either mice or humans. In TRAMP mice, urinary 8-oxodG levels were elevated with increasing age (p < 0.0001) but not changed by the presence of prostate tumours. In conclusion, the LC/MS/MS SRM methods described here are ideally suited for the accurate determination of 8-oxodG and creatinine in urine samples from both clinical and pre-clinical studies.
    • Determination of endogenous and exogenously derived N7-(2-hydroxyethyl)guanine adducts in ethylene oxide-treated rats.

      Marsden, Debbie A.; Jones, Donald J.L.; Lamb, John H.; Tompkins, Elaine M.; Farmer, Peter B.; Brown, Karen (2007-02)
      Ethylene oxide (EO) is one of the most widely used intermediates in the chemical industry. It is also formed endogenously as a result of cytochrome P450-mediated metabolism of ethylene, which is ubiquitous in the environment. Additionally, ethylene is generated in vivo during normal physiological processes such as methionine oxidation and lipid peroxidation; therefore, humans are continually exposed to EO. EO is classed by the IARC as carcinogenic to humans and reacts with DNA, primarily forming N7-(2-hydroxyethyl)guanine adducts (N7-HEG), which can be used as biomarkers of exposure and potential cancer risk. To assess the risks to humans associated with occupational exposure to low EO concentrations, it is necessary to establish the relative contribution of DNA damage arising from endogenous and exogenously derived EO. Using a newly developed highly sensitive LC-MS/MS assay with selected reaction monitoring that offers a limit of detection of 0.1 fmol of N7-HEG on column, we have established background levels of N7-HEG (1.1-3.5 adducts/10(8) nucleotides) in tissues of rats. Following intraperitoneal administration of a single dose or three daily doses of EO (0.01-1.0 mg/kg), N7-HEG adducts generally increased with dose, except at the lowest concentration where total N7-HEG levels were no different to that detected in control animals, indicating that any increase was negligible as compared to the endogenous damage already present. In the 3 day study, the kinetics of adduct removal were also investigated and in comparing N7-HEG formation in the two studies, DNA damage did not appear to accumulate with repeated administration.
    • Development and application of biomarker methods in molecular environmental epidemiology for the detection of exposure to polycyclic aromatic hydrocarbons.

      Kovács, Katalin (University of Pécs, 2012, 2012)
      My research aimed at the further development of the 32P-postlabelling method by which, whilst maintaining the adduct-labelling efficiency, the amount of the radioisotope per sample can be reduced, and thereby the sample processing capacity of the laboratory is increased. The regular and the modified 32P-postlabelling methods were used for comparative determinations of the levels of DNA adducts from various types of DNA samples, i.e. BPDE-DNA adduct standard, DNA samples from MCF-7 cell line treated with B[a]P, DNA samples from human non-tumorous peripheral lung tissue, human peripheral blood lymphocytes and human buffy coat samples. In the modified 32P-postlabelling method, the final reaction volume of the radio-labelling was reduced with an evaporation-to-dryness step to one-third of the volume that was used in the regular method. This facilitated the reduction of the amount of [γ-32P]ATP substrate from 50 μCi per sample – depending on the DNS isolation method – by 50% for the Qiagen-isolated samples (i.e., 25 μCi per sample) and by 80% for the DNA samples isolated with the classic phenol extraction procedure (i.e.,10 μCi) for both experimental and human samples. The newly-developed BPDE-DNA direct sandwich chemiluminescence immunoassay (BPDE-DNA SCIA) for the determination of PAH-DNA adducts has been published in 2012. Whereas in the earlier competitive immunoassays the signal to be measured is coupled to the DNA adducts, in the SCIA the chemiluminescent signal is coupled to the non-adducted nucleotides. In SCIA, the strong end-point signal derives from the many orders of magnitude difference between the number of the non-adducted and the adducted mononucleotides. The limit of detection of the method is ~ 1.5 adducts/109 nucleotides from 5 μg DNA sample. For the validation of BPDE-DNA SCIA, I performed comparative measurements between the immunoassay and the 32P-postlabelling method. The DNA samples were obtained from MCF 7 cells treated with B[a]P, from the liver of mice, which were treated in vivo with several doses of B[a]P, B[b]F, and DB[a,h]A, respectively, and from human maternal peripheral blood and newborn cord blood samples. For the B[a]P-DNA adduct levels measured by SCIA and 32P-postlabelling from the MCF 7 cells, the ratio between the adduct values was about 0.5. For the animal samples, the adduct levels were several times lower by the immunoassay than by the 32P-postlabelling method (the ratios were ≈1:5 for B[a]P, ≈ 1:30 for B[b]F and ≈ 1:5 for DB[a,h]A). All the same, there was a very strong, highly significant positive correlation between the DNA adduct measurements of the dose-response curves by SCIA and 32P-postlabelling for each PAH compound (r = 0.87-0.99). For the human samples, the ratio between the SCIA and 32P-postlabelling values was approximately 1:10, but there was not correlation between the data-pairs measured by the two methods. For the human samples, the lack of correlation between the two methods may be explained by the different efficiency of detection of different structural types of DNA adducts that are derived from complex human environmental exposure.
    • Development and validation of a direct sandwich chemiluminescence immunoassay for measuring DNA adducts of benzo[a]pyrene and other polycyclic aromatic hydrocarbons.

      Georgiadis, Panagiotis; Kovacs, Katalin; Kaila, Stella; Makedonopoulou, Paraskevi; Anna, Livia; Poirier, Miriam C.; Knudsen, Lisbeth E.; Schoket, Bernadette; Kyrtopoulos, Soterios A. (2012-05-18)
      We have developed and validated a sandwich chemiluminescence immunoassay (SCIA) which measures polycyclic aromatic hydrocarbon (PAH)-DNA adducts combining high throughput and adequate sensitivity, appropriate for evaluation of adduct levels in human population studies. Fragmented DNA is incubated with rabbit antiserum elicited against DNA modified with r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and subsequently trapped by goat anti-rabbit IgG bound to a solid surface. Anti-single-stranded (ss) DNA antibodies binds in a quantity proportional to the adduct levels and is detected by chemiluminescence. The BPDE-DNA SCIA has a limit of detection of 3 adducts per 10(9) nucleotides with 5 μg DNA per well. We have validated the BPDE-DNA SCIA using DNA modified in vitro, DNA from benzo[a]pyrene (BP)-exposed cultured cells and mice. The levels of adduct measured by SCIA were lower (30-60%) than levels of bulky DNA adducts measured in the same samples by (32)P-postlabelling. The BPDE-DNA SCIA also detected adducts produced in vivo by PAHs other than BP. When blood DNA samples from maternal/infant pairs were assayed by BPDE-DNA SCIA, the adduct levels obtained were significantly correlated. However, there was no correlation between (32)P-postlabelling and SCIA values for the same samples. The SCIA can be extended to any DNA adduct and is expected to provide, when fully automated, a valuable high-throughput approach in large-scale population studies.
    • Development and validation of a new, sensitive immunochemical assay for O⁶-methylguanine in DNA and its application in a population study.

      Georgiadis, Panagiotis; Kaila, Stella; Makedonopoulou, Paraskevi; Fthenou, Eleni; Chatzi, Leda; Pletsa, Vasiliki; Kyrtopoulos, Soterios A. (2011-01)
      Investigations of the presence of the precarcinogenic DNA adduct O⁶-methylguanine (O6-meG) in humans and its association with exposure or cancer risk have been hindered by the absence of analytic methods of adequate sensitivity and throughput. We report the development, validation, and application of an ELISA-type assay for O6-meG appropriate for large-scale population studies.
    • Development of a liquid chromatography-electrospray ionization tandem mass spectrometry method for detecting oxaliplatin-DNA intrastrand cross-links in biological samples.

      Le Pla, Rachel C.; Ritchie, Kenneth J.; Henderson, Colin J.; Wolf, C. Roland; Harrington, Chris F.; Farmer, Peter B. (2007-08)
      Cellular resistance, both intrinsic and acquired, poses a problem in the effectiveness of platinum-based chemotherapy. The cytotoxic activity of Pt-based chemotherapeutic agents is derived from their ability to react with cellular DNA. Oxaliplatin binds to the N7 position of the purine DNA bases, forming mainly intrastrand cross-links between either two adjacent guanines (GG), an adjacent adenine and guanine (AG), or two guanines separated by an unmodified nucleotide (GNG). We report the development of a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for measuring GG and AG intrastrand cross-links formed by oxaliplatin. The limits of detection for GG-oxPt and AG-oxPt were 23 and 19 adducts per 10 (8) nucleotides, respectively. We compare the formation and persistence of intrastrand cross-links between wild-type and glutathione transferase P null mice (GSTP null) treated with oxaliplatin. No significant difference was observed in the level of intrastrand cross-links formed by oxaliplatin between the mouse strains in liver, kidney, and lung DNA. Adduct levels were greatest in liver and lowest in lung tissue.
    • Development of a novel site-specific mutagenesis assay using MALDI-ToF MS (SSMA-MS).

      McLuckie, Keith I.E.; Lamb, John H.; Sandhu, Jatinderpal K.; Pearson, Helen L.; Brown, Karen; Farmer, Peter B.; Jones, Donald J.L. (2006)
      We have developed and validated a novel site-specific mutagenesis assay, termed SSMA-MS, which incorporates MALDI-ToF mass spectrometry (MALDI-MS) analysis as a means of determining the mutations induced by a single DNA adduct. The assay involves ligating an adducted deoxyoligonucleotide into supF containing pSP189 plasmid. The plasmid is transfected into human Ad293 kidney cells allowing replication and therefore repair or a mutagenic event to occur. Escherichia coli indicator bacteria are transformed with recovered plasmid and plasmids containing the insert are identified colormetrically, as they behave as frameshift mutations. The plasmid is then amplified and digested using a restriction cocktail of Mbo11 and Mnl1 to yield 12 bp deoxyoligonucleotides, which are characterized by MALDI-MS. MALDI-MS takes advantage of the difference in molecular weight between bases to identify any induced mutations. This analysis method therefore provides qualitative and quantitative information regarding the type and frequency of mutations induced. This assay was developed and validated using an O(6)-methyl-2'-deoxyguanosine adduct, which induced the expected GC-->AT substitutions, when replicated in human or bacterial cells. This approach can be applied to the study of any DNA adduct in any biologically relevant gene sequence (e.g. p53) in human cells and would be particularly amenable to high-throughput analysis.
    • Dietary intake of meat and meat-derived heterocyclic aromatic amines and their correlation with DNA adducts in female breast tissue.

      Rohrmann, Sabine; Lukas Jung, Sea-Uck; Linseisen, Jakob; Pfau, Wolfgang (2009-03)
      It was the aim of this study to examine the association of the consumption of meat in general, meat prepared by different cooking methods and the dietary intake of heterocyclic aromatic amines (HCA) with the level of DNA adducts in the breast tissue of women undergoing reduction mammoplasty. Dietary intake of meat and HCA were assessed via questionnaire in 44 women undergoing reduction mammoplasty. DNA adduct analysis in breast tissue was performed by (32)P-postlabelling analysis. Spearman rank correlation coefficients (r) were calculated to examine the association of meat consumption and dietary HCA intake with tissue DNA adduct levels. A median DNA adduct level of 18.45 (interquartile range 12.81-25.65) per 10(9) nucleotides in breast tissue was observed; median HCA intake was 40.43 ng/day (interquartile range 19.55-102.33 ng/day). Total HCA intake (r = 0.33, P = 0.03), consumption of fried meat (r = 0.39, P = 0.01), beef (r = 0.32, P = 0.03) and processed meat (r = 0.51, P = 0.0004) were statistically significantly correlated with the level of DNA adducts in breast tissue. The detected DNA adducts could not be confirmed to be specific HCA-derived DNA adducts by comparison with external standards, using the (32)P-postlabelling assay. We observed strong correlations of dietary HCA intake and consumption of fried and processed meat with DNA adduct levels in breast tissue of 44 women. Since the detected DNA adducts were not necessarily specific only for HCA, it is possible that HCA intake is a surrogate of other genotoxic substances, such as polycyclic aromatic hydrocarbons, in meat prepared at high temperatures.
    • Dietary intake of vitamin K and risk of prostate cancer in the Heidelberg cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC-Heidelberg).

      Nimptsch, Katharina; Rohrmann, Sabine; Linseisen, Jakob (2008-04)
      BACKGROUND: Anticarcinogenic activities of vitamin K have been observed in various cancer cell lines, including prostate cancer cells. Epidemiologic studies linking dietary intake of vitamin K with the development of prostate cancer have not yet been conducted. OBJECTIVE: We evaluated the association between dietary intake of phylloquinone (vitamin K1) and menaquinones (vitamin K2) and total and advanced prostate cancer in the Heidelberg cohort of the European Prospective Investigation into Cancer and Nutrition. DESIGN: At baseline, habitual dietary intake was assessed by means of a food-frequency questionnaire. Dietary intake of phylloquinone and menaquinones (MK-4-14) was estimated by using previously published HPLC-based food-content data. Multivariate-adjusted relative risks of total and advanced prostate cancer in relation to intakes of phylloquinone and menaquinones were calculated in 11 319 men by means of Cox proportional hazards regression. RESULTS: During a mean follow-up time of 8.6 y, 268 incident cases of prostate cancer, including 113 advanced cases, were identified. We observed a nonsignificant inverse association between total prostate cancer and total menaquinone intake [multivariate relative risk (highest compared with lowest quartile): 0.65; 95% CI: 0.39, 1.06]. The association was stronger for advanced prostate cancer (0.37; 0.16, 0.88; P for trend = 0.03). Menaquinones from dairy products had a stronger inverse association with advanced prostate cancer than did menaquinones from meat. Phylloquinone intake was unrelated to prostate cancer incidence (1.02; 0.70, 1.48). CONCLUSIONS: Our results suggest an inverse association between the intake of menaquinones, but not that of phylloquinone, and prostate cancer. Further studies of dietary vitamin K and prostate cancer are warranted.
    • Dietary vitamins, polyphenols, selenium and probiotics: biomarkers of exposure and mechanism of anticarcinogenic action.

      The Nofer Institute of Occupational Medicine, 2007-04
      The major aim of this review was to summarize the state-of-the-art in use of biomarkers for some anticarcinogenic food components and to identify knowledge gaps, especially those of relevance for the partners of the NoE ECNIS and its contacts. Since this is a vast area, only certain selected topics, as outlined below, are considered in detail. The important links found between use of a substance as a biomarker and its mechanisms of action led to a further aim, that of reviewing the mechanisms of action of some of the most promising anticarcinogenic compounds.
    • Different mechanisms involved in apoptosis following exposure to benzo[a]pyrene in F258 and Hepa1c1c7 cells.

      Holme, Jorn A.; Gorria, Morgane; Arlt, Volker M.; Ovrebo, Steinar; Solhaug, Anita; Tekpli, Xavier; Landvik, Nina E.; Huc, Laurence; Fardel, Olivier; Lagadic-Gossmann, Dominique (2007-04-05)
      The present study compares and elucidates possible mechanisms why B[a]P induces different cell signals and triggers apparently different apoptotic pathways in two rather similar cell lines (hepatic epithelial cells of rodents). The rate and maximal capacity of metabolic activation, as measured by the formation of B[a]P-tetrols and B[a]P-DNA adducts, was much higher in mouse hepatoma Hepa1c1c7 cells than in rat liver epithelial F258 cells due to a higher induced level of cyp1a1. B[a]P increased intracellular pH in both cell lines, but this change modulated the apoptotic process only in F258 cells. In Hepa1c1c7 cells reactive oxygen species (ROS) production appeared to be a consequence of toxicity, unlike F258 cells in which it was an initial event. The increased mitochondrial membrane potential found in F258 cells was not observed in Hepa1c1c7 cells. Surprisingly, F258 cells cultured at low cell density were somewhat more sensitive to low (50nM) B[a]P concentrations than Hepa1c1c7 cells. This could be explained partly by metabolic differences at low B[a]P concentrations. In contrast to the Hepa1c1c7 model, no activation of cell survival signals including p-Akt, p-ERK1/2 and no clear inactivation of pro-apoptotic Bad was observed in the F258 model following exposure to B[a]P. Another important difference between the two cell lines was related to the role of Bax and cytochrome c. In Hepa1c1c7 cells, B[a]P exposure resulted in a "classical" translocation of Bax to the mitochondria and release of cytochrome c, whereas in F258 cells no intracellular translocation of these two proteins was seen. These results suggest that the rate of metabolism of B[a]P and type of reactive metabolites formed influence the resulting balance of pro-apoptotic and anti-apoptotic cell signaling, and hence the mechanisms involved in cell death and the chances of more permanent genetic damage.
    • Differential protection by human glutathione S-transferase P1 against cytotoxicity of benzo[a]pyrene, dibenzo[a,l]pyrene, or their dihydrodiol metabolites, in bi-transgenic cell lines that co-express rat versus human cytochrome P4501A1.

      Kabler, Sandra L.; Seidel, Albrecht; Jacob, Juergen; Doehmer, Johannes; Morrow, Charles S.; Townsend, Alan J. (2009-05-15)
      Polycyclic aromatic hydrocarbons (PAHs) are activated by cytochrome P450 (CYP) isozymes, and a subset of the reactive metabolites generated is detoxified via conjugation with glutathione (GSH) by specific glutathione S-transferases (GSTs). We have used V79MZ cells stably transfected with either human or rat cytochrome P4501A1 (CYP1A1), alone or in combination with human GSTP1 (hGSTP1), to examine the dynamics of activation versus detoxification of benzo[a]pyrene (B[a]P), dibenzo[a,l]pyrene (DB[a,l]P), and their dihydrodiol metabolites. The cytotoxicity of B[a]P or DB[a,l]P was 9-11-fold greater in cells expressing human, as compared to rat CYP1A1, despite similar enzymatic activities. Co-expression of the hGSTP1 with the hCYP1A1 conferred 16-fold resistance to B[a]P cytotoxicity, compared to only 2.5-fold resistance when hGSTP1 was co-expressed with rat CYP1A1. The lower B[a]P cytotoxicity in the cells expressing rat CYP1A1, and weaker protection by hGSTP1 co-expression in these cells, were attributable to the much lower fraction of B[a]P metabolism via formation of the 7,8-dihydrodiol intermediate by the rat CYP1A1 compared to hCYP1A1. Resistance to the DB[a,l]P cytotoxicity conferred by hGSTP1 expression was also greater in cells co-expressing hCYP1A1 (7-fold) as compared to cells co-expressing rCYP1A1 (<2-fold). Resistance to B[a]P conferred by hGSTP1 was closely correlated with the activity level in two clonal transfectant lines with a 3-fold difference in hGSTP1-1 specific activity. Depletion of GSH to 20% of control levels via pretreatment with the de novo GSH biosynthesis inhibitor buthionine sulfoximine reduced the protection against B[a]P cytotoxicity by hGSTP1 from 16-fold to 5-fold, indicating that catalysis of conjugation with GSH, rather than binding or other effects, is responsible for the resistance. The cytotoxicity of the dihydrodiol intermediates of B[a]P or DB[a,l]P was much greater, and similar in cell lines expressing either human or rat CYP1A1. Again, however, the protection conferred by hGSTP1 co-expression was 2-5-fold greater in cells with hCYP1A1 than with rCYP1A1 expression. These results indicate that GST expression can effectively limit cytotoxicity following activation of B[a]P by human or rat CYP1A1, but is less effective as a defense against exposure of cells to the intermediate metabolite B[a]P-7,8-dihydrodiol.
    • DNA adduct formation and oxidative stress from the carcinogenic urban air pollutant 3-nitrobenzanthrone and its isomer 2-nitrobenzanthrone, in vitro and in vivo.

      Nagy, Eszter; Adachi, Shuichi; Takamura-Enya, Takeji; Zeisig, Magnus; Moller, Lennart (2007-03)
      The carcinogenic vehicle emission product 3-nitrobenzanthrone (3-NBA) is known to rearrange in the atmosphere to the isomer 2-nitrobenzanthrone (2-NBA), which exists in 70-fold higher concentration in ambient air. The genotoxicity of 2-NBA and 3-NBA was studied both in vitro (human cell lines A549 and HepG2) and in vivo (F344 female rats intra-tracheally administered 5 mg/kg body weight of 3-NBA) models, using the (32)P-HPLC and the single-cell gel electrophoresis (Comet assay) methods. In vitro, also the parent compound benzanthrone (BA) and the metabolite 3-aminobenzanthrone (3-ABA) were evaluated. 3-NBA gave highest levels of DNA adducts in the two cell lines, but significantly higher in HepG2 (relative adduct level approximately 500 adducts/10(8) normal nucleotides), whereas 2-NBA formed about one-third and one-twentieth of the DNA adduct amount in A549 and HepG2 cells, respectively. 3-ABA formed only minute amounts of DNA adducts and only in the A549 cells, whereas BA did not give rise to any detectable levels. The DNA adduct patterns from 3-NBA were similar between the two model systems, but differed somewhat for 2-NBA. The oxidative stress induced by BA was almost as high as what was observed for 3-NBA and 3-ABA in both cell lines, and 2-NBA induced lowest level of oxidative stress. The oxidative stress and DNA adduct level, in whole blood, was significantly increased by 3-NBA but not by 2-NBA. However, 2-NBA showed similar toxicity to 3-NBA, with respect to DNA adduct formation in vivo, hence it is important to further study 2-NBA as a potential contributor to health risk. While DNA adduct level in the 3-NBA-exposed animals reached a peak around 1 and 2 days after instillation, 2-NBA-treated animals showed a tendency towards a continuing increase at the end of the study.
    • DNA adducts and cancer risk in prospective studies: a pooled analysis and a meta-analysis.

      Veglia, Fabrizio; Loft, Steffen; Matullo, Giuseppe; Peluso, Marco; Munnia, Armelle; Perera, Frederica; Phillips, David H.; Tang, Deliang; Autrup, Herman; Raaschou-Nielsen, Ole; et al. (2008-05)
      Bulky DNA adducts are biomarkers of exposure to aromatic compounds and of the ability of the individual to metabolically activate carcinogens and to repair DNA damage. Their ability to predict cancer onset is uncertain. We have performed a pooled analysis of three prospective studies on cancer risk in which bulky DNA adducts have been measured in blood samples collected from healthy subjects (N = 1947; average follow-up 51-137 months). In addition, we have performed a meta-analysis by identifying all articles on the same subject published up to the end of 2006, including case-control studies. In the pooled analysis, a weakly statistically significant increase in the risk of lung cancer was apparent (14% per unit standard deviation change in adduct levels, 95% confidence interval 1-28%; using the weighted mean difference method, 0.15 SD, units higher adducts in cases than in controls). The association was evident only in current smokers and was absent in former smokers. Also the meta-analysis, which included both lung and bladder cancers, showed a statistically significant association in current smokers, whereas the results in never smokers were equivocal; in former smokers, no association was detected. The results of our pooled and meta-analyses suggest that bulky DNA adducts are associated with lung cancer arising in current smokers after a follow-up of several years.
    • DNA adducts in normal colonic mucosa from healthy controls and patients with colon polyps and colorectal carcinomas.

      Jonsson, Charlotte; Stal, Per; Sjoqvist, Urban; Akerlund, Jan-Erik; Lofberg, Robert; Moller, Lennart (2010-09)
      Colon cancer is a multistage process where adenomatous polyps developing in a normal mucosa may further progress to neoplasia. DNA adducts are biomarkers linked to exposure to carcinogenic compounds, tumour formation and clinically observed cancer. Such DNA adducts have been detected in the mucosa of colon cancer patients. The aim of this study was to investigate whether there are differences in DNA adduct levels and patterns in mucosa from patients with colon cancer, polyps and non-cancerous controls and whether some DNA adducts could be markers for colon cancer development. Human colonic biopsies were collected from healthy controls (n = 10), polyp patients (n = 22) (from both normal and polyp tissue) and colon cancer patients (n = 32) (from both tumour tissue and adjacent normal mucosa). In 150 tissues specimens (when small amount of tissue, the same type of tissues were pooled from each patient), DNA adduct levels and patterns were analysed by the (32)P-high-performance liquid chromatography method. There were no significant difference in the total levels of DNA adducts between any of the groups. Levels of two single DNA adducts were decreased in mucosa adjacent to tumours as compared to mucosa from healthy controls. One DNA adduct was found only in tumour tissue and adjacent mucosa from the colon cancer patients. A food derived, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-related DNA adduct was detected in 106 of the 150 tissues analysed, but in similar levels in tissues from controls, polyp patients or cancer patients. In conclusion, three individual DNA adducts may be interesting candidates for further evaluation of their possible role as biomarkers in human carcinogenesis. Furthermore, a food-derived PhIP-related adduct contributes to the general DNA adduct pattern in most individuals, indicating a minor role of this adduct in human colon carcinogenesis.
    • DNA and protein adducts in human tissues resulting from exposure to tobacco smoke.

      Phillips, David H.; Venitt, Stan (2012-12-15)
      Tobacco smoke contains a variety of genotoxic carcinogens that form adducts with DNA and protein in the tissues of smokers. Not only are these biochemical events relevant to the carcinogenic process, but the detection of adducts provides a means of monitoring exposure to tobacco smoke. Characterization of smoking-related adducts has shed light on the mechanisms of smoking-related diseases and many different types of smoking-derived DNA and protein adducts have been identified. Such approaches also reveal the potential harm of environmental tobacco smoke (ETS) to nonsmokers, infants and children. Because the majority of tobacco-smoke carcinogens are not exclusive to this source of exposure, studies comparing smokers and nonsmokers may be confounded by other environmental sources. Nevertheless, certain DNA and protein adducts have been validated as biomarkers of exposure to tobacco smoke, with continuing applications in the study of ETS exposures, cancer prevention and tobacco product legislation. Our article is a review of the literature on smoking-related adducts in human tissues published since 2002.
    • DNA damage and acute toxicity caused by the urban air pollutant 3-nitrobenzanthrone in rats: characterization of DNA adducts in eight different tissues and organs with synthesized standards.

      Nagy, Eszter; Adachi, Shuichi; Takamura-Enya, Takeji; Zeisig, Magnus; Moller, Lennart (2006-08)
      3-Nitrobenzanthrone (3-NBA) is an urban air pollutant and rat lung carcinogen that is among the most potent mutagens yet tested in the Salmonella reversion assay. In the present study, 1 mg 3-NBA was administered orally to female F344 rats and DNA adduct formation was examined in liver, lung, kidney and five sections of the gastrointestinal (GI) tract at 6 hr, and 1, 2, 3, 5, and 10 days after administration. The DNA adduct patterns, analyzed by (32)P-postlabelling followed by HPLC separation, were similar in all tissues and organs. Five of the adduct peaks cochromatographed with synthesized DNA adduct standards. Three of these unequivocally determined standards, dGp-C8-N-ABA, dGp-N2-C2-ABA, and dAp-N6-C2-ABA, were of the nonacetylated type, suggesting that at least part of the pathway for activation of 3-NBA proceeds through O-acetylation of the hydroxylamine intermediate. The two other DNA adduct standards, dGp-C8-C2-N-Ac-ABA, and dGp-N2-C2-N-Ac-ABA, were of the acetylated type, but there was some ambiguity in the characterization of these DNA adducts, since they varied inconsistently between samples and they also aligned with peaks found in controls. At 6 hr after treatment, the level of DNA adducts was highest in glandular stomach (relative adduct labeling (RAL), approximately 70 adducts/10(8) normal nucleotides (NN)); adduct levels in this organ decreased at 24 hr, but increased afterwards. DNA adduct levels in the majority of organs were characterized by an early increase (from 6 hr to 3 days), which was followed by a decrease at 5 days and a maximum level 10 days after administration (RAL approximately 120 adducts/10(8) NN for the lung, kidney and glandular stomach, approximately 80 adducts/10(8) NN for the forestomach and ceacum, and approximately 40 adducts/10(8) NN for the liver, small intestine, and colon). This pattern was consistent with pathological observations during autopsy showing high levels of tissue damage in the GI tract; the tissue damage included hemorrhages, loss of villous surface structure in the small intestine, as well as intestine fragility and oedema of the adipose tissue around the GI-tract. Tissue damage decreased and DNA adduct levels increased at 10 days after administration. These observations suggest that 3-NBA not only exerts acute toxic effects, but that the bioavailability is affected by storage in tissues and later becomes available, resulting in the increased DNA adduct levels at the later time points of collection.
    • DNA damage and cytotoxicity in type II lung epithelial (A549) cell cultures after exposure to diesel exhaust and urban street particles.

      Danielsen, Pernille Hogh; Loft, Steffen; Moller, Peter; I (2008)
      ABSTRACT: BACKGROUND: Exposure to air pollution particles has been acknowledged to be associated with excess generation of oxidative damage to DNA in experimental model systems and humans. The use of standard reference material (SRM), such as SRM1650 and SRM2975, is advantageous because experiments can be reproduced independently, but exposure to such samples may not mimic the effects observed after exposure to authentic air pollution particles. This study was designed to compare the DNA oxidizing effects of authentic street particles with SRM1650 and SRM2975. The authentic street particles were collected at a traffic intensive road in Copenhagen, Denmark. RESULTS: All of the particles generated strand breaks and oxidized purines in A549 lung epithelial cells in a dose-dependent manner and there were no overt differences in their potency. The exposures also yielded dose-dependent increase of cytotoxicity (as lactate dehydrogenase release) and reduced colony forming ability with slightly stronger cytotoxicity of SRM1650 than of the other particles. In contrast, only the authentic street particles were able to generate 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in calf thymus DNA, which might be due to the much higher level of transition metals. CONCLUSION: Authentic street particles and SRMs differ in their ability to oxidize DNA in a cell-free environment, whereas cell culture experiments indicate that the particle preparations elicit a similar alteration of the level of DNA damage and small differences in cytotoxicity. Although it cannot be ruled out that SRMs and authentic street particles might elicit different effects in animal experimental models, this study indicates that on the cellular level, SRM1650 and SRM2975 are suitable surrogate samples for the study of authentic street particles.