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The genotoxic potential of extractable organic matter (EOM) associated with the respirable particulate matter (PM10 microm) of atmospheric pollution has been determined in three European cities--Prague (Czech Republic, two monitoring sites, Libus and Smíchov), Kosice (Slovak Republic) and Sofia (Bulgaria) using the alkaline single-cell gel electrophoresis (the comet assay). The EOM samples were extracted by dichloromethane from ambient airborne particles collected daily (24 h intervals) during 3-month sampling periods in winter and summer seasons. The human metabolically competent cell line Hep G2 was used as a test system and benzo[a]pyrene (BaP), a known carcinogen, was applied as a positive control (internal standard) in each electrophoretic run. Two-hour exposure of Hep G2 cells to equivalent EOM concentrations ranging from 5 to 150 microg EOM/ml resulted in a linear dose-dependent increase of DNA migration (r0.9, P0.01). A less significant dose-response (r = 0.61) was only induced by the EOM sample from the locality Prague-Libus (PRG-LB) in the winter. Generally, a 1.5 to four-fold increase of DNA strand breaks over the background control level was determined in EOM-exposed cells. In order to compare the genotoxic potential of individual EOMs, a mathematical model was used to correct the 'real' data. No substantial location- or season-related differences were found in EOM genotoxicity (EOM microg/ml), except for the EOM sample from Sofia, collected in the summer. This EOM sample induced a nearly two-fold lower level of DNA damage in comparison with other EOMs. On the other hand, clear statistically significant location- and season-related differences (P0.001) in ambient air genotoxicity were determined when the EOM quantity per cubic meter of air (microg/m3) was taken into account. In that case, the genotoxicity of winter air pollution was six- to 10-fold higher in comparison with summer air. The air pollution genotoxicity in individual localities rose during the winter season in the order: PRG-LBKosicePrague-Smíchov (PRG-SM)Sofia, while during the summer season the highest ambient air genotoxicity was revealed in the locality Prague-Smíchov and approximately equal air pollution genotoxicity was determined among localities Prague-Libus, Kosice and Sofia (PRG-LB approximately Kosice approximately SofiaPRG-SM). The greatest overall air pollution genotoxicity was determined in the locality Sofia during the winter season. In a time course study to evaluate the kinetics of DNA strand break rejoining it was shown that the level of DNA strand breaks in EOM-exposed cells has returned to near the background level within 24 h after the treatment.

Indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM) are promising cancer chemopreventive agents in rodent models, but there is a paucity of data on their pharmacokinetics and tissue disposition. The disposition of I3C and its acid condensation products, DIM, [2-(indol-3-ylmethyl)-indol-3-yl]indol-3-ylmethane (LTr1), indolo[3,2b]carbazole (ICZ) and 1-(3-hydroxymethyl)-indolyl-3-indolylmethane (HI-IM) was studied, after oral administration of I3C (250 mg/kg) to female CD-1 mice. Blood, liver, kidney, lung, heart, and brain were collected between 0.25 and 24 h after administration and the plasma and tissue concentrations of I3C and its derivatives determined by high-performance liquid chromotography. I3C was rapidly absorbed, distributed, and eliminated from plasma and tissues, falling below the limit of detection by 1 h. Highest concentrations of I3C were detected in the liver where levels were approximately 6-fold higher than those in the plasma. Levels of DIM, LTr1, and HI-IM were much lower, although they persisted in plasma and tissues for considerably longer. DIM and HI-IM were still present in the liver 24 h after I3C administration. Tissue levels of DIM and LTr1 were found to be in equilibrium with plasma at almost every time point measured. In addition to acid condensation products of I3C, a major oxidative metabolite (indole-3-carboxylic acid) and a minor oxidative metabolite (indole-3-carboxaldehyde) were detected in plasma of mice after oral administration of I3C. ICZ was also tentatively identified in the liver of these mice. This study shows for the first time that, after oral administration to mice, I3C, in addition to its acid condensation products, is absorbed from the gut and distributed systemically into a number of well-perfused tissues, thus allowing the possibility for some pharmacological activity of the parent compound in vivo.

Determination of the interaction products (adducts) of a carcinogen with DNA or protein indicates the amount of genotoxic material that has reached the tissue under study and provides a valuable biomarker of exposure for molecular epidemiological studies. DNA adducts may also give further information with regard to the mutagenic significance of the exposure. The sensitivity and applicability of the analytical methods for the detection and quantification of carcinogen adducts has greatly increased in recent years, and DNA damage levels as low as one adduct per 108 nucleotides can now routinely be measured. The discovery of many types of endogenously-produced damage of DNA and protein has demonstrated previously unsuspected sources of genotoxicity, the biological consequences of which are so far not known.

Styrene is widely used in the production of various plastics, synthetic rubber and resins. The aim of this study was to evaluate if individual polymorphisms in xenobiotic metabolizing enzymes, related with the metabolic fate of styrene, could modify individual susceptibility to the possible genotoxic effects of the styrene exposure. Twenty-eight reinforced plastic workers and 28 control subjects were studied. In the selected population the urinary styrene metabolites mandelic (MA) and phenylglyoxylic (PGA) acids were quantified, sister chromatid exchanges (SCE) and micronuclei (MN) were assessed in peripheral lymphocytes and all the subjects were genotyped for GSTM1 , GSTT1 (gene deletions), GSTP1 (codon 105 ile ⇒ val), EPHX1 (codons 113 tyr ⇒ his and 139 his ⇒ arg) and CYP2E1 ( Dra I polymorphism in intron 6). The results obtained showed a significant difference between the levels of SCE, but not in MN levels, in exposed workers as compared with the control group. The GSTP1 and CYP2E1 individual genotypes modulate the baseline levels of SCE that are lower in non-wild type individuals for both polymorphisms. The GSTM1 null individuals with low levels of exposure have significantly higher urinary levels of MA+PGA. The present data seem to suggest that apart from the methodology usually used for monitoring populations occupationally exposed to styrene (urinary metabolites and biomarkers of early biological effects) the analysis of individual genotypes associated with the metabolic fate of styrene should also be carried out in order to evaluate the individual genetic susceptibility of exposed populations.

Resveratrol (trans-3,5,4'-trihydroxystilbene) is a naturally occurring polyphenol with cancer chemopreventive properties in preclinical models of carcinogenesis, including those of colorectal cancer. Recently, a variety of analogues of resveratrol have been synthesised and investigated in in vitro assays. One analogue, 3,4,5,4'-tetramethoxystilbene (DMU 212), showed preferential growth-inhibitory and proapoptotic properties in transformed cells, when compared with their untransformed counterparts. As part of a chemoprevention drug development programme, the pharmacokinetic properties of DMU 212 were compared with those of resveratrol in the plasma, liver, kidney, lung, heart, brain and small intestinal and colonic mucosa of mice. DMU 212 or resveratrol (240 mg kg(-1)) were administered intragastrically, and drug concentrations were measured by HPLC. Metabolites were characterised by cochromatography with authentic reference compounds and were identified by mass spectrometry. The ratios of area of plasma or tissue concentration vs time curves of resveratrol over DMU 212 (AUC(res)/AUC(DMU212)) for the plasma, liver, small intestinal and colonic mucosa were 3.5, 5, 0.1 and 0.15, respectively. Thus, resveratrol afforded significantly higher levels than DMU 212 in the plasma and liver, while DMU 212 exhibited superior availability compared to resveratrol in the small intestine and colon. Resveratrol was metabolised to its sulphate or glucuronate conjugates, while DMU 212 underwent metabolic hydroxylation or single and double O-demethylation. DMU 212 and resveratrol inhibited the growth of human-derived colon cancer cells HCA-7 and HT-29 in vitro with IC(50) values of between 6 and 26 microM. In the light of the superior levels achieved in the gastrointestinal tract after the administration of DMU 212, when compared to resveratrol, the results provide a good rationale to evaluate DMU 212 as a colorectal cancer chemopreventive agent.

Exposure to high levels of environmental air pollution is known to be associated with an increased carcinogenic risk. The individual contribution to this risk derived from specific carcinogenic chemicals within the complex mixture of air pollution is less certain, but may be explored by the use of molecular epidemiological techniques. Measurements of biomarkers of exposure, of effect and of susceptibility provide information of potential benefit for epidemiological and cancer risk assessment. The application of such techniques has been mostly concerned in the past with the carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) that are associated with particulate matter in air pollution, and has showed clear evidence of genotoxic effects, such as DNA adducts, chromosome aberrations (CA) and ras oncogene overexpression, in environmentally exposed Czech and Polish populations. We are currently extending these studies by an investigation of populations exposed to environmental pollution in three European countries, Czech Republic, Slovak Republic and Bulgaria. This pays particular attention to PAHs, but also investigates the extent of radically induced (oxidative) DNA damage in the exposed populations. Policemen, bus drivers and controls, who carried personal monitors to determine their exposures to PAHs have been studied, and blood and urine were collected. Antioxidant and dietary status were assessed in these populations. Stationary monitors were also used for ambient air monitoring. Amongst the parameters studied in the biological samples were: (a) exposure biomarkers, such

In this communication we present data on S-phenylcysteine (SPC) in human serum albumin in an Estonian cohort. A total of 112 human samples were collected from workers of a benzene processing plant in Kohtla-Jarve, Estonia and another 56 samples from nearby countryside residents. Albumin was isolated using a sequential precipitation method designed for low volume plasma samples. The adducted cysteine derivatives were cleaved off from the albumin and subsequently derivatized to phenyl trifluorothioacetate (PTTA) derivatives. Derivatized adducts were subjected to GC/MS analysis utilizing negative chemical ionization and a selected ion monitoring (SIM) technique. The detection limit for the PTTA adduct was 0.2 pmol g−1 albumin and the limit of quantification was 2 pmol g−1 albumin. The method utilized deuterated SPC as an internal standard. The analysis revealed that adduct levels were also high in the population that was not occupationally exposed to benzene. The reason for this is unknown, but may cause pro...

The aim of this study was to use DNA adducts levels, detected by 32P-postlabelling, as a biomarker to assess human exposure to polycyclic aromatic hydrocarbons (PAHs) from a coke oven plant and explore the possible association between CYP1A1 MspI, GSTP1, GSTM1 and GSTT1 genotypes, and smoking status on bulky DNA adduct formation. A large amount of inter-individual variation in adduct level was observed among workers with the same job and the same smoking habits. No significant differences were observed in DNA adduct levels between the coke oven workers and control group. Smokers in the control group had significantly higher DNA adducts than the non-smokers in the same group (35.13+/-21.11 versus 11.18+/-8.00, per 10(8) nucleotides, P=0.003). In this group, the correlation between the level of DNA adducts and the cigarettes smoked was strongly significant (r=0.70, P

Stable and specific biomacromolecular adducts can be used to measure in vivo doses of reactive compounds. An LC/MS–MS method to measure adducts from the benzo[a]pyrene (BP) metabolite (±)-anti-BP-7,8-diol-9,10-epoxide ((±)-anti-BPDE) to His146 in serum albumin (SA), earlier evaluated on in vitro alkylated human SA, was tested for its applicability to mouse. It was shown that (+)-anti-BPDE form BPDE-His adducts to mouse SA. The method was applied to samples from BP-exposed mice (100 mg/kg of body weight for 1, 3, 7 and 28 days). BPDE-His in SA was close to the limit of quantification and showed the highest level (13 fmol/mg) 3 days after exposure. The level was 400 times lower (calculated per gram macromolecule) than earlier measured level of BPDE-adduct to deoxyguanosine (dG) in DNA in the livers. The relative rate of formation of adducts from BPDE with His in SA and with dG in DNA was investigated. Quantification by LC/MS–MS of the adducts in human blood alkylated in vitro with (±)-anti-BPDE showed a 1850 times higher level of BPDE-dG compared to BPDE-His. The specific and stable BPDE-adducts to His in SA are potential biomarkers of in vivo dose of BPDE, though this requires a considerable improved analytical sensitivity of the LC/MS–MS method.

Epidemiologic studies indicate that prolonged exposure to high pollution levels is associated with increased risk of cancer, especially lung cancer. However, under conditions of moderate or low air pollution, epidemiologic evidence does not permit reliable conclusions. Biomarker-based population studies may serve as complementary tools providing a better understanding of the relative contribution of ambient atmospheric pollution to the overall genotoxic burden suffered by city dwellers. However, past efforts to apply biomarkers to studies of low levels exposure to urban air pollution have given inconclusive results, partly because of the absence of adequate data on personal exposure, covering a time-window which is appropriate for the biomarkers being examined, as well as a battery of biomarkers reflecting different stages of the carcinogenic process. In the present paper, the potential of biomarker-based population studies to aid the assessment of the genotoxic and carcinogenic effects of urban air pollution is reviewed by reference to the achievements and limitations of earlier reported studies. The design and methodology adopted in a recently completed large-scale population study, carried out in the context of the European Union Environment and Climate Programme, known by the short name of AULIS project, is discussed and descriptive statistics of the main findings of the project are presented. These findings indicate that for cohorts suffering moderate-to-low exposures to airborne particulate-bound polycyclic aromatic hydrocarbons (PAHs), no simple correlation with biomarkers of genotoxicity existed and suggest that additional factors made a significant contribution to the overall genotoxic burden.

It has been widely reported that the Harderian gland, present in most vertebrates, accumulates high levels of porphyrins, particularly protoporphyrin. The present study describes the extraction, identification and characterization of a group of hitherto unreported protoporphyrin glycoconjugates in the rat Harderian gland using HPLC, capillary electrophoresis, on-line HPLC/electrospray ionization MS and tandem MS. The major glycoconjugate was identified as protoporphyrin-1-O-acyl β-xyloside with a smaller amount of protoporphyrin-1-O-acyl β-glucoside also detected. In the Harderian glands studied, 50-70% of the porphyrins present were in the form of protoporphyrin glycoconjugates. This is the first reported occurrence of glycoconjugates of porphyrins in Nature and suggests that previous studies have wrongly identified the major porphyrin in the Harderian gland as the unconjugated protoporphyrin.

An assay has been developed to determine S-phenylcysteine (SPC) in globin as a potential biomarker for exposure to benzene. The sensitivity of the assay is less than 20 pmol SPC g(-1) globin. Following acidic hydrolysis of the protein, the modified amino acid is purified by reverse phase cartridge chromatography and HPLC, prior to conversion to the tert-butyldimethylsilyl derivative and GC-MS selected ion recording. Quantitation is achieved using the internal standard [(2)H5]-SPC, and calibration lines were established using a synthetic peptide Leu-His-SPC-Asp-Lys. Control human globin was found to contain ca 30 pmol SPC g(-1) globin in two populations. The source of the apparent background level of SPC is unknown.

The formation of DNA adducts by the covalent binding of genotoxic chemicals to DNA represents a valuable marker for assessing exposure to carcinogens but as yet the role of DNA adducts as a biomarker of carcinogenic susceptibility still needs to be clearly ascertained. To address this question an animal study was instigated using mice (SWR (high), BALB/c (intermediate) and C57BL/6J (low)) varying in their susceptibility to lung carcinogenesis. Groups of animals from each strain were dosed with a single intraperitoneal injection of saline or N -nitrosodiethylamine (NDEA) at 15 or 90 mg kg(-1) body weight. Lung and liver tissues were removed at different time points following dosing. Further groups of mice dosed with the same regime had urine samples collected 24 h post dosing and were then left up to 18 months to allow for the development of tumours. Immunoslot-blot analysis was used for the determination of N-7 ethylguanine (N-7EtG) and O(6) ethylguanine (O(6)EtG) adduct levels in the DNA from the tissues and gas chromatography-mass spectrometry (GC-MS) was used to determine N-3 ethyladenine (N-3EtA) adduct levels in the urine samples. Levels of alkyltransferase (ATase) were also determined in the tissues. The results showed that the DNA adduct levels and persistence were similar across the three strains of mice following dosing with 15 and 90 mg kg(-1) NDEA. High levels of adducts were observed in the urine of the BALB/c strain, implying an increased metabolic or repair capacity in this strain. However there were no differences in the levels of ATase in the lung and liver of the three strains of mice following dosing with 15 mg kg(-1) NDEA. The incidence of tumours in C57BL/6J mice was lower compared with the other two strains and showed a dose dependent increase. The results from this study show that the differences in susceptibility to lung carcinogenesis between the three strains of mice do not appear to be linked to the formation of the two adducts detected. These results imply that dosing with NDEA resulted in toxicity which may have led to cell death and induction of tumours by compensatory cell proliferation. Although these results do not allow decisive conclusions to be drawn concerning the relationship between total levels of DNA adducts and differences in carcinogenic susceptibility for the three strains of mice it is clear that the increased presence of a DNA adduct in the target tissue increases the likelihood of tumour development.

Incidence rates for non-Hodgkin's lymphoma (NHL) have been rising throughout the world for several decades, and no convincing explanation exists for the majority of this increase. The commonest subtypes of NHL have no well-defined aetiological factors but lymphoma development has been linked with exposure to a variety of chemicals, including nitrates, pesticides, herbicides, and solvents. Benzene, a solvent and important constituent of petrochemical products, is a potent lymphomagen in experimental animals and high-dose exposure in humans is associated with both acute myeloid leukaemia and NHL. Much current interest centres on the possibility that environmental benzene exposure in the general public may underlie a proportion of the increase in NHL. Seventy per cent of benzene exposure in the environment is derived from vehicle exhaust emissions, whose increase has closely paralleled the rise in frequency of the disease. Mathematical modelling has been used to calculate an acceptable concentration of benzene in air based on risk estimates derived from industrial exposure, but the recommended target concentration in the U.K. of 1 ppb is regularly exceeded in urban locations. Detailed investigation of the health effects of low-level benzene exposure awaits an accurate assay for quantifying long-term human exposure. The (32)P post-labelling technique for the detection of toxin-specific DNA adducts is extremely sensitive and has been applied in the biomonitoring of exposure to a number of carcinogens, but benzene-DNA adducts have to date proved elusive of detection.

A study employing several biomarkers of styrene exposure and genotoxicity was carried out in a group of lamination (reinforced plastic) workers and controls, who had been repeatedly sampled during a 3-year period. Special attention will be paid to the last sampling (S.VI), reported here for the first time. Styrene concentration in the breathing zone, monitored by personal dosimeters, and urinary mandelic acid (MA) were measured as indicators of external exposure. Blood samples were assayed for styrene-specific O6-guanine adducts in DNA, N-terminal valine adducts of styrene in haemoglobin, DNA single-strand breaks (SSB), determined by use of the single cell gel electrophoresis (Comet) assay), and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutant frequencies (MF) in T-lymphocytes. O6-styrene guanine adduct levels were significantly higher in the exposed group (5.9 +/- 4.9 adducts/10(8) dNp) as compared to laboratory controls (0.7 +/- 0.8 adducts/10(8) dNp; P = 0.001). DNA adduct levels significantly correlated with haemoglobin adducts, SSB parameters and years of employment. Styrene-induced N-terminal valine adducts were detected in the lamination workers (1.7 +/- 1.1 pmol/g globin), but not in the control group (detection limit 0.1 pmol/g globin). N-terminal valine adducts correlated strongly with external exposure indicators, DNA adducts and HPRT MF. No significant correlation was found with SSB parameters. A statistically significant difference in HPRT MF was observed between the laminators (22.3 +/- 10.6/10(6)) and laboratory controls (14.2 +/- 6.5/10(6), P = 0.039). HPRT MF in the laminators significantly correlated with styrene concentration in air, MA and haemoglobin adducts, as well as with years of employment and age of the employees. No significant difference (P = 0.450) in MF between the laminators and the factory controls was observed. Surprisingly, we detected differences in MF between sexes. When data from all measurements were combined, women showed higher MF (geometric mean 15.4 vs. 11.2 in men, P = 0.020). The styrene-exposed group exhibited significantly higher SSB parameters (tail moment (TM), tail length (TL) and the percentage of DNA in the tail (TP)) than the control group (P < 0.001). SSB parameters correlated with indicators of external exposure and with O6-styrene guanine adducts. No significant correlation was found between SSB parameters and haemoglobin adducts or HPRT MF. The data encompassing biomarkers from repeated measurements of the same population over a 3-year period are discussed with respect to the mechanisms of genotoxic effects of styrene and the interrelationship of individual biomarkers.

Malondialdehyde (MDA) is a product of lipid peroxidation and prostaglandin biosynthesis. It is mutagenic and carcinogenic and the major adduct formed by reaction with DNA, a highly fluorescent pyrimidopurinone (M 1 -dG), has been detected in healthy human liver and leukocyte DNA. Analytical methods used so far for the detection of M 1 -dG have not been applied to a large number of individuals or variety of samples. Often, only a few μg of DNA from human tissues are available for analysis and a very sensitive assay is needed in order to detect background levels of M 1 -dG in very small amounts of DNA. In this paper, the development of an immunoslot blot (ISB) assay for the measurement of M 1 -dG in 1 μg of DNA is described. The limit of detection of the assay is 2.5 adducts per 10 8 bases. A series of human samples were analysed and levels of 5.6-9.5 (n = 8) and 3.1-64.3 (n = 42) of M 1 -dG per 10 8 normal bases were detected in white blood cell and gastric biopsy DNA, respectively. Results on four human samples were compared with those obtained using an HPLC/ 32 P-post-labelling (HPLC/PPL) method previously developed and indicated a high correlation between M 1 -dG levels measured by the two assays. The advantages of ISB over other assays including HPLC/PPL, such as the possibility of analysing 1 μg DNA/sample and the fact that it is less time-consuming and laborious, means that it can be more easily used for routine analysis of a large number of samples in biomonitoring studies.

The DNA adduct levels in total white blood cells (WBC) and lymphocytes (LYM) isolated from the blood of the same individuals were evaluated using the -postlabelling assay for bulky aromatic adducts. In this study, 68 male coke oven workers and 56 machine workers as a matched control were enrolled. Personal monitors were used to evaluate exposure to eight carcinogenic PAHs, including B[a]P, during an 8-h working shift. The exposure among coke oven workers ranged widely from 0.6 to 547 μg/m3 and from 2 to 62,107 ng/m3, for carcinogenic PAHs and B[a]P, respectively. The respective values in controls were from 0.07–1.64 μg/m3 and from 1–63 ng/m3. A significant correlation between WBC– and LYM–DNA adduct levels was found (r=0.591, P

A ring-test was organised between three laboratories using different versions of the modified Edman degradation technique for the gas chromatographic-mass spectrometric determination of N-terminal valine adducts of styrene 7,8-oxide. The analyses were performed on a sample of human haemoglobin reacted in vitro with styrene 7,8-oxide and on a set of five haemoglobin samples from mice dosed by i.p. injection of styrene. Strong correlations between the haemoglobin adduct determinations of the different laboratories were observed. However, covariance analysis revealed different slopes for the dose-response curves, indicating differences for the calibration of the reference globin or reference peptide.

(R)-(2,3,4,5,6-Pentadeuterophenyl)oxirane, 5 was synthesised with an overall yield of 6.3% from benzene-d 6 . Friedel-Crafts acylation of benzene-d 6 with methyl oxalylchloride gave methyl 2-oxo-2-(2,3,4,5,6-pentadeuterophenyl)acetate, I that was chirally reduced (Saccharomyces cerevisiae) to methyl (R)-2-hydroxy-2-(2,3,4,5,6-pentadeuterophenyl)acetate (methyl (R)-d,-mandelic acid), 2. Reduction of 2 with lithium aluminium hydride gave (R)-(2,3,4,5,6-pentadeuterophenyl)ethane-1,2-diol, 3. Tosylation of 3 and subsequent treatment with potassium hydroxide assisted cyclisation to 5.

Postlabeling can be one of the most sensitive methods for the measurement of DNA adducts. However, for the determination of alkylated adducts, essential requirements are standards which must be fully chemically characterized. In order to develop a postlabeling assay for monitoring exposure to genotoxic ethylating agents, the reaction of diethyl sulfate with 2'-deoxynucleoside 3'- and 5'-monophosphates was examined. The adducts generated were fully characterized using HPLC, electrospray tandem mass spectrometry, UV, and postlabeling. The major product was the phosphodiester derived from alkylation of the phosphate, and alkylation of the base occurred to a lesser extent. The phosphodiester standard, 2'-deoxyguanosine 3'-(mono-O-ethyl phosphate) (3'Et-pdG), was used to develop a postlabeling assay for the detection of this adduct in DNA samples. Since alkylated phosphodiesters in DNA are not susceptible to the actions of micrococcal nuclease and calf spleen phosphodiesterase, they can be obtained as alkylated phosphodiester dinucleosides from DNA. Nuclease P1 was used as enhancement step which allowed the separation of these adducted phosphotriesters from the unmodified nucleotides by HPLC. Subsequent hydrolysis of the phosphotriester dinucleosides in alkali yielded phosphodiesters, including 3'Et-pdG, which was efficiently postlabeled. This approach was shown to be capable of detecting this adduct in liver DNA from mice treated intraperitoneally with N-nitrosodiethylamine.

In January 1993 the oil tanker Braer ran aground in the Shetland Islands, Scotland. Approximately 80,000 tons of crude oil were released. Exceptionally high winds caused extensive pollution and exposure of the local population to crude oil. We describe the study which was immediately set in place to examine the exposed population for evidence of genotoxic exposure. Blood samples were taken and primary DNA damage was measured in the mononuclear cell fraction by the butanol modification of the 32P-postlabelling method. Mutation was measured at the hprt locus in T lymphocytes. No evidence of genotoxicity was obtained for either end point, but nevertheless, we believe that useful lessons were learnt, which should be incorporated into the design of future studies: (1) A rapid response is essential, and even if sufficient funds are not immediately available, it is still worth attempting to obtain samples quickly and use cryopreservation, also to attempt to estimate exposure. (2) Adequate numbers of volunteers must be sought, together with enough controls, not just to allow meaningful analysis but to overcome loss of samples and failure of things to go according to plan. (3) Points concerning laboratory practice include: (i) samples should be coded, (ii) clearly defined and proven protocols should be used, (iii) irreplaceable samples should not be used for method development, (iv) should a problem become apparent during the study, work on such samples should cease immediately until the problem is solved, (v) all critical experimental components should be pretested against a laboratory standard. (4) The study design should include replicate experiments to monitor experimental variability and reproducibility, as well as internal standards and cryopreserved "in house" samples. Care must be taken that samples from any one exposure group are spread between a number of independent experiments and that each experiment includes samples from a number of exposure groups. (5) A computerised data base should be maintained with full details of experimental variables, donor attributes, and raw data so that any contribution of experimental artefacts to "outlier" results can be monitored. (6) Because of the nature of the statistical variation for many environmental genotoxicity end points, only a large-scale study is likely to be capable of yielding useful information.

The European Food Safety Authority (EFSA) was asked by the European Commission to deliver a scientific opinion on ergot alkaloids (EAs) in food and feed. EAs are produced by several members within the fungal orders of Hypocreales and Eurotiales. In Europe, Claviceps purpurea is the most widespread Claviceps species within the Hypocreales. A total of 20 558 analytical results for EAs in 1 716 food, 496 feed and 67 unprocessed grain samples were considered in this opinion. Based on the EAs identified in sclerotia of C. purpurea, and recent literature data, the EFSA Panel on Contaminants in the Food Chain (CONTAM Panel) based its risk assessment on the main C. purpurea EAs, namely ergometrine, ergotamine, ergosine, ergocristine, ergocryptine (which is a mixture of a- and ß- isomers), ergocornine, and the corresponding –inine epimers. The CONTAM Panel performed estimates of both chronic and acute exposure for various age groups across European countries. A BMDL10 of 0.33 mg/kg b.w. per day was calculated for the incidence of tail muscular atrophy in a 13-week rat feeding study of ergotamine. This effect was considered representative of the vasoconstrictive effects of EAs and provided a suitable reference point for establishment of a group acute reference dose of 1 µg/kg body weight (b.w.) and a group tolerable daily intake of 0.6 µg/kg b.w. per day. The Panel concluded that whilst the available data do not indicate a concern for any population subgroup, the dietary exposure estimates relate to a limited number of food groups and a possible unknown contribution from other foods cannot be discounted. Estimates of exposure for livestock based on example diets and levels of EAs in cereal grains reported suggest that under normal conditions the risk of toxicosis is low.

Arylisocyanates are important intermediates in the chemical industry. Amongst the main damage after low levels of isocyanate exposure are lung sensitization and asthma. Protein adducts of isocyanates might be involved in the aetiology of sensitization reactions. Blood protein adducts are used as dosimeters for modifications of macromolecules in the target organs where the disease develops. To develop methods for the quantitation of protein adducts we reacted 4 methylphenyl isocyanate 4MPI with the tripeptide valyl glycyl glycine and with single amino acids yielding N 4 methylphenyl carbamoyl L valyl glycyl glycine 4MPI Val Gly Gly, N 4 methylphenyl carbamoyl L valine 4MPI Val, N 4 methylphenyl carbamoyl L aspartic acid 4MPI Asp, N acetyl S 4 methylphenyl carbamoyl L cysteine 4MPI AcCys, N acetyl N 4 methylphenyl carbamoyl lysine 4MPI AcLys, N acetyl O 4 methylphenyl carbamoyl tyrosine 4MPI AcTyr and N acetyl O 4 methylphenyl carbamoyl D,L serine 4MPI AcSer. The hydrolysis of the adducts was tested under acidic and basic conditions, to obtain the maximum yield of 4 methylaniline 4MA. The isocyanates were hydrolysed for 1 h, 3h and 24h at 100 C with 6 M HCl in and or 0.1 M NaOH at room temperature, following methods applied for the analyses of biological samples of arylisocyanate exposed workers. In addition, we applied a new protocol: the adducts were hydrolyzed for 1-24 h in 0.3 M NaOH at 100 C. The hydrolysates were analysed using HPLC with UV detection and quantified against the internal standard, 4 fluoroaniline or 4 chloroaniline. 4MA was obtained with the best yields using 0.3M NaOH; after 24 h all amino acid adducts were cleaved under these conditions. Acid hydrolysis of 4MPI Val and 4MPI Asp yielded the respective hydantoins 3 4 methylphenyl 5 isopropyl 1,3 imidazoline 2,4 dione and 2 1 4 methylphenyl 2,5 dioxoperhydro 4 imidazolyl acetic acid. For future studies, we propose to hydrolyse biological samples with 0.3 M NaOH at 100 C to release the maximum amount of 4MA from the adducts. However, in biological samples from workers, hydrolysable adducts can also result from arylamine exposure. Therefore, we propose to analyse the N terminal adducts of isocyanates with blood protein to distinguish between arylamine and arylisocyanate exposure.

Atylamines and nitroarenes are very important environmental and occupational pollutants. Genotoxic effects of arylamines are believed to be initiated by the formation of DNA adducts. DNA adducts of arylamines have been found in experimental animals and in exposed humans, and are predominantly formed with the carbon 8 of 2'-deoxyguanosine. Reference standards are necessary to develop methods for the quantification of DNA-adducts. Therefore, we have synthesized the 2'-deoxyguanosin-8-yI adducts of 2-methylaniline, 2-chloroaniline, 4-chloroaniline, 2,4dimethylaniline, and 2,6-dimethylaniline. The products were characterized by (1)H-NMR, (13)C-NMR, MS and UV. The corresponding 2'-deoxyguanosine-3' -monophosphate adducts were synthesized for the quantification of DNA adducts by the (32)P-postlabelling technique. A GC-MS method was developed for the analysis of the new adducts as an alternative to the (32)P-postlabelling. DNA was spiked with the synthesized adducts and treated with 0.3 m NaOH overnight at 110 °C in the presence of a deuterated internal standard. We observed up to 80% recovery from about 1 adduct in 10(8) to 1 in 10(5) nucleotides.

Human exposure to ethylene oxide and acrylonitrile may be monitored by determination of the products that these electrophilic compounds form with the amino terminal of hemoglobin. The procedure involves a modified Edman degradation, using the reagent pentafluorophenyl isothiocyanate, which cleaves the adducted N-terminal amino acid from the protein chain as a substituted pentafluorophenyl thiohydantoin. This may then be quantitated using selected ion monitoring gas chromatography-mass spectrometry (GC-MS). To overcome problems arising from the lack of an ideal internal standard for this analysis, we have now synthesized adducts of ethylene oxide and acrylonitrile with the N-terminal tripeptide of the α-chain of human globin (Val-Leu-Ser). These adducts should behave analogously to the protein adduct in the Edman degradation. The adducts were characterized by nuclear magnetic resonance and fast atom bombardment and electrospray MS and tandem MS. Additionally, an analogous stable isotope-labeled standard was synthesized for the acrylonitrile adduct using a dg-labeled tripeptide. Quantitative calibration lines for analysis of the adducts have been established and the validity of the assays established by the detection of acrylonitrile adducts in the globin of acrylonitrile-exposed workers and of cigarette smokers. © 1996 Wiley-Liss, Inc.

A coordinated study was carried out on the development, evaluation and application of biomonitoring procedures for populations exposed to environmental genotoxic pollutants. The procedures used involved both direct measurement of DNA or protein damage (adducts) and assessment of secondary biological effects (mutation and cytogenetic damage). Adduct detection at the level of DNA or protein (haemoglobin) was carried out by 32P-postlabelling, immunochemical, HPLC or mass spectrometric methods. Urinary excretion products resulting from DNA damage were also estimated (immunochemical assay, mass spectrometry). The measurement of adducts was focused on those from genotoxicants that result from petrochemical combustion or processing, e.g. low-molecular-weight alkylating agents, PAHs and compounds that cause oxidative DNA damage. Cytogenetic analysis of lymphocytes was undertaken (micronuclei, chromosome aberrations and sister chromatid exchanges) and mutation frequency was estimated at a number of loci including the hprt gene and genes involved in cancer development. Blood and urine samples from individuals exposed to urban pollution were collected. Populations exposed through occupational or medical sources to larger amounts of some of the genotoxic compounds present in the environmental samples were used as positive controls for the environmentally exposed population. Samples from rural areas were used as negative controls. The project has led to new, more sensitive and more selective approaches for detecting carcinogen-induced damage to DNA and proteins, and subsequent biological effects. These methods were validated with the occupational exposures, which showed evidence of DNA and/or protein and/or chromosome damage in workers in a coke oven plant, garage workers exposed to diesel exhaust and workers exposed to ethylene oxide in a sterilization plant. Dose response and adduct repair were studied for methylated adducts in patients treated with methylating cytostatic drugs. The biomonitoring methods have also demonstrated their potential for detecting environmental exposure to genotoxic compounds in nine groups of non-smoking individuals, 32P-postlabelling of DNA adducts being shown to have the greatest sensitivity. Chemicals/CAS: Antineoplastic Agents, Alkylating; Blood Proteins; Carcinogens, Environmental; DNA Adducts; Epichlorohydrin, 106-89-8; Ethylene Oxide, 75-21-8; Methylene Chloride, 75-09-2; Mutagens; Nitrogen Oxides; Petroleum; Polycyclic Hydrocarbons, Aromatic; Styrene, 100-42-5; Styrenes

For genotoxic carcinogens, exposure assessment may be achieved by measurements of the extent of covalent interaction (adduct formation) that has occurred between the carcinogen and macromolecules such as DNA, haemoglobin and albumin. Adducts for many carcinogens have been found in supposedly unexposed populations. This signifies either that endogenous processes contribute to this DNA/protein modification, or that there are exogenous exposures to these carcinogens that were not previously recognised. Notable examples where 'background' genotoxic modification has been found include damage caused by low molecular weight alkylating agents and hydroxyl radicals. The significance of the existence of these adducts to genotoxic risk is as yet unknown.

OBJECTIVE--The aim was to assess the significance of two biomarkers; antibody to benzo(a)pyrene DNA adducts and concentration of hydroxyethylvaline haemoglobin adducts in samples from a well studied group of coke oven workers. As a measure of exposure we have used 1-hydroxypyrene in urine. METHODS--Urine and blood samples were collected from coke oven workers and a control group. Samples from coke oven plant workers were collected in January and June. 1-Hydroxypyrene was measured in urine by high performance liquid chromatography (HPLC), antibodies to benzo(a)pyrene DNA adducts were measured by ELISA and hydroxyethylvaline haemoglobin adducts were measured by gas chromatography-mass spectrometry (GC-MS). RESULTS--Mean urinary 1-hydroxypyrene in samples from coke oven workers varied from 1.11 to 5.53 umol/mol creatinine and 0.14 umol/mol creatinine in the control group. Workers at the top side had the highest values of urinary 1-hydroxypyrene. Antibody to benzo(a)pyrene DNA adducts did not correlate with either 1-hydroxypyrene nor length of work at the coke oven plant. But antibody concentration in samples collected in January was predictive of the concentration in samples collected in June. A small non-significant increase in hydroxyethylvaline haemoglobin adducts was found in samples from coke oven workers relative to the control group when comparing smokers and nonsmokers separately. CONCLUSION--1-Hydroxypyrene correlates well with exposure groups based on job description. Antibodies to benzo(a)-pyrene DNA adducts was related to people and not exposure. Work at a coke oven plant might lead to increased hydroxyethylvaline haemoglobin adducts.

The European Food Safety Authority (EFSA) was asked by the European Commission to deliver a scientific opinion on ergot alkaloids (EAs) in food and feed. EAs are produced by several members within the fungal orders of Hypocreales and Eurotiales. In Europe, Claviceps purpurea is the most widespread Claviceps species within the Hypocreales. A total of 20 558 analytical results for EAs in 1 716 food, 496 feed and 67 unprocessed grain samples were considered in this opinion. Based on the EAs identified in sclerotia of C. purpurea, and recent literature data, the EFSA Panel on Contaminants in the Food Chain (CONTAM Panel) based its risk assessment on the main C. purpurea EAs, namely ergometrine, ergotamine, ergosine, ergocristine, ergocryptine (which is a mixture of α- and β- isomers), ergocornine, and the corresponding –inine epimers. The CONTAM Panel performed estimates of both chronic and acute exposure for various age groups across European countries. A BMDL10 of 0.33 mg/kg b.w. per day was calculated for the incidence of tail muscular atrophy in a 13-week rat feeding study of ergotamine. This effect was considered representative of the vasoconstrictive effects of EAs and provided a suitable reference point for establishment of a group acute reference dose of 1 μg/kg body weight (b.w.) and a group tolerable daily intake of 0.6 μg/kg b.w. per day. The Panel concluded that whilst the available data do not indicate a concern for any population subgroup, the dietary exposure estimates relate to a limited number of food groups and a possible unknown contribution from other foods cannot be discounted. Estimates of exposure for livestock based on example diets and levels of EAs in cereal grains reported suggest that under normal conditions the risk of toxicosis is low.

Styrene is metabolized to styrene oxide, a direct-acting mutagen and carcinogen. Styrene oxide reacts with DNA mainly at the N-7 position in guanine, but also at other sites and with other bases. Substitution occurs at both the alpha- and beta-positions of the styrene molecule. Experiments with radiolabeled styrene and styrene oxide demonstrate that both have a low level of DNA binding activity in experimental animals. 32P-Postlabeling studies have demonstrated the potential of the technique to detect styrene-DNA adducts. Styrene oxide alkylates several nucleophilic sites in proteins, particularly cysteine sulfydryl, histidine imidazole, lysine amino, aspartic, and glutamic carboxylic groups, and the N-terminal position. In experimental animals, styrene oxide treatment results in cysteine adducts in hemoglobin and albumin, valine adducts in hemoglobin, and carboxylic acid adducts in hemoglobin. The extent of alkylation is low compared with that produced by ethylene oxide. The available evidence indicates, therefore, that styrene and styrene oxide have low DNA and protein binding activities in vivo. There is preliminary evidence for the presence of DNA adducts and for adducts in hemoglobin and albumin in blood cells of styrene-exposed workers. Nevertheless, the applicability and sensitivity of DNA and protein adduct detection methods for monitoring human exposure to styrene remain to be determined.

The European Food Safety Authority (EFSA) was asked by the European Commission to deliver a scientific opinion on pyrrolizidine alkaloids (PA) in food and feed. PAs are toxins exclusively biosynthesised by plants. To date, approximately 600 different PAs are known. Results for 13,280 bulk honey and 1324 retail honey samples were provided to EFSA by one Member State and 351 feed samples were provided by a second Member State. The EFSA Panel on Contaminants in the Food Chain (CONTAM Panel) performed estimates of both acute and chronic exposure to PAs through honey for three different age groups. Although there might be other sources of PA exposure, due to lack of data the CONTAM Panel was not able to quantify dietary exposure from food other than honey. A number of PAs were identified as being of particular importance for food and feed. Based on the present knowledge of metabolism, activation, DNA adduct‐formation, genotoxicity and carcinogenicity, the CONTAM Panel concluded that 1,2–unsaturated PAs may act as genotoxic carcinogens in humans. Therefore, the CONTAM Panel decided to apply the Margin of Exposure (MOE) approach. A benchmark dose lower confidence limit for a 10 % excess cancer risk (BMDL10) of 70 µg/kg b.w. per day for induction of liver haemangiosarcomas by lasiocarpine in male rats was calculated as the reference point for comparison with the estimated dietary exposure. The CONTAM Panel concluded that there is a possible health concern for those toddlers and children who are high consumers of honey. There is generally a low risk of PA poisoning in livestock and companion animals in the EU as most PA poisonings reported recently are due to accidental exposure.

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.

The ability to non-invasively assess DNA oxidation and its repair, has significant utility in large-scale, population-based studies. Such studies could include the assessments of: the efficacy of antioxidant intervention strategies, pathological roles of DNA oxidation in various disease states and population or interindividual differences in antioxidant defence and DNA repair. The most popular method, to non-invasively assess oxidative insult to the genome is by the analysis of urine for 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), using chromatographic techniques or immunoassay procedures. The provenance of extracellular 8-oxodG remains a subject for debate. However, previous studies have shown that factors, such as diet and cell death, do not appear to contribute to extracellular 8-oxodG, leaving processes, such as the repair of DNA and/or the 2'-deoxyribonucleotide pool, as the sole source of endogenous 8-oxodG. The method in this chapter describes a non-invasive approach for assessing oxidative stress, via the efficient extraction of urinary 8-oxodG using a validated solid-phase extraction procedure. Subsequent analysis by liquid chromatography-tandem mass spectrometry provides the advantages of sensitivity, internal standardisation, and robust peak identification, and is widely considered to be the "gold standard".

This article is being simultaneously published in 2011 in PLoS Medicine, Journal of Clinical Epidemiology, Preventive Medicine, Mutagenesis, Journal of Epidemiology and Community Health, European Journal of Epidemiology and European Journal of Clinical Investigation. Reproduced by permission of the authors. The authors jointly hold the copyright of this article. 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. This paper is part of an activity sponsored by the ECNIS network (EC grant FOOD-CT-2005-513943) (www.ecnis.org). We acknowledge the contribution of Dan Segerbäck, Jim Vaught, Soterios Kyrtopoulos, Franz Oesch, Jelle Vlaanderen, and Jouni Jaakkola to the discussion of an earlier version of the paper.

3-Chloroperbenzoic acid-17O,18O was prepared from 3-chlorobenzoyl chloride and hydrogen peroxide-17O,18O (obtained by subjecting water-17O,18O. Nitrobenzene-15N was reduced to aniline-15N, which was oxidised by 3-chloroperbenzoic acid to nitrosobenzene-15N. 15N and 17O NMR data are given for the corresponding labelled nitrosobenzene.

In contrast with fractions from control animals, an aldehyde reductase, which catalyses the reduction of aflatoxin B1-dihydrodiol, in the dialdehyde form at physiological pH values, to aflatoxin B1-dialcohol, is expressed in cytosolic fractions prepared from rat livers bearing pre-neoplastic lesions, or following treatment with the anti-oxidant ethoxyquin. This expression parallels the development of resistance to the toxin. Unlike the aflatoxin B1-dihydrodiol, the dialcohol does not undergo binding to protein. This enzyme activity could play a mechanistic role in hepatocarcinogenesis and chemoprotection in the rat. Correlated n.m.r. and m.s. spectra are provided in Supplementary Publication SUP 50171 (3 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1993) 289, 9.

Monitoring exposure to alkylating agents may be achieved by quantitatively determining the adduct levels formed with nucleic acids and/or proteins. One of the most significant results arising from the application of this approach has been the discovery in control populations of "background" levels of alkylated nucleic acid bases or alkylated proteins, in particular hemoglobin (Hb). In the case of Hb, a wide variety of such adducts have been detected and quantitated by mass spectrometric techniques, with methylated, 2-carboxyethylated, and 2-hydroxyethylated modifications being most abundant. Although the source of these alkylation products is unknown, both endogenous and exogenous sources may be proposed. We have recently confirmed the presence of the N-terminal hydroxyethylvaline adduct in control human Hb using tandem mass spectrometry (MS-MS) and have now established background levels using GC-MS in more than 70 samples. Smoking raises the levels of the adduct up to 10-fold and occupational exposure to ethylene oxide up to 300-fold. Background levels of alkylated nucleic acids may be studied by analysis of N7-alkylated guanine or N3-alkylated adenine, which are excised from nucleic acids after their formation and are excreted in urine. Although the presence of some of these urinary constituents may be accounted for by their natural occurrence in RNA or diet, the endogenous or exogenous source of others is unknown. Quantitative methods using MS-MS have now been developed for five of the observed urinary alkylguanines [N7-methyl-, N2-methyl-, N2-dimethyl-, N7-(2-hydroxyethyl)-, and N2-ethylguanine].(ABSTRACT TRUNCATED AT 250 WORDS)

The ability to non-invasively assess DNA oxidation and its repair, has significant utility in large-scale, population-based studies. Such studies could include the assessments of: the efficacy of antioxidant intervention strategies, pathological roles of DNA oxidation in various disease states and population or interindividual differences in antioxidant defence and DNA repair. The most popular method, to non-invasively assess oxidative insult to the genome is by the analysis of urine for 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), using chromatographic techniques or immunoassay procedures. The provenance of extracellular 8-oxodG remains a subject for debate. However, previous studies have shown that factors, such as diet and cell death, do not appear to contribute to extracellular 8-oxodG, leaving processes, such as the repair of DNA and/or the 2'-deoxyribonucleotide pool, as the sole source of endogenous 8-oxodG. The method in this chapter describes a non-invasive approach for assessing oxidative stress, via the efficient extraction of urinary 8-oxodG using a validated solid-phase extraction procedure. Subsequent analysis by liquid chromatography-tandem mass spectrometry provides the advantages of sensitivity, internal standardisation, and robust peak identification, and is widely considered to be the "gold standard".

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.

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. The authors acknowledge financial support from the European Union Network of Excellence ECNIS, Type B project (Environmental Cancer Risk, Nutrition and Individual Susceptibility, www.ecnis.org, Contract No. FOOD-CT-2005-513943)

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 (P0.05) 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.

A significant proportion of human cancers overexpress DNA polymerase beta (Pol beta), the major DNA polymerase involved in base excision repair. The underlying mechanism and biological consequences of overexpression of this protein are unknown. We examined whether Pol beta, expressed at levels found in tumor cells, is involved in the repair of DNA damage induced by oxaliplatin treatment and whether the expression status of this protein alters the sensitivity of cells to oxaliplatin. DNA damage induced by oxaliplatin treatment of HCT116 and HT29 colon cancer cells was observed to be associated with the stabilization of Pol beta protein on chromatin. In comparison with HCT116 colon cancer cells, isogenic oxaliplatin-resistant (HCT-OR) cells were found to have higher constitutive levels of Pol beta protein, faster in vitro repair of a DNA substrate containing a single nucleotide gap and faster repair of 1,2-GG oxaliplatin adduct levels in cells. In HCT-OR cells, small interfering RNA knockdown of Pol beta delayed the repair of oxaliplatin-induced DNA damage. In a different model system, Pol beta-deficient fibroblasts were less able to repair 1,2-GG oxaliplatin adducts and were hypersensitive to oxaliplatin treatment compared with isogenic Pol beta-expressing cells. Consistent with previous studies, Pol beta-deficient mouse fibroblasts were not hypersensitive to cisplatin treatment. These data provide the first link between oxaliplatin sensitivity and DNA repair involving Pol beta. They demonstrate that Pol beta modulates the sensitivity of cells to oxaliplatin treatment.

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 4.7nmol/L (adjusted for specific weight; range 1.6-13, corresponding to 1.7mug/g creatinine, range 0.57-4.8) and of total urinary As metabolites (U-As) 290mug/L (range 94-720; 380mug/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. (Less)

OBJECTIVES: There is some evidence that dietary components that are rich in antioxidant and vitamins are inversely associated with DNA adduct levels induced by environmental carcinogens such as polycyclic aromatic hydrocarbons, although the epidemiologic data are inconsistent. This study addresses the association between vitamins, DNA adducts and smoking. METHODS: A combined analysis of individual data on the association between bulky DNA adducts and dietary vitamins was conducted. A Medline search was performed to identify studies on healthy subjects in which smoking and vitamins intake information were available, and bulky DNA adducts were measured in peripheral blood with 32P-postlabelling. Eight published studies met the eligibility criteria, and individual data from 7 data sets including 2758 subjects were obtained. GSTM1 and GSTT1 were also available on all the subjects. RESULTS: Vitamin E was inversely significantly associated with DNA adducts after adjustment for possible confounding factors. Vitamins A and C were not independent predictors of DNA adducts. A stratified analysis showed that vitamin A had a significant inverse association with DNA adducts in ever smokers only. CONCLUSIONS: This result is relevant to planning any future chemo-preventive interventions directed to high risk subgroups of the population, for cancer prevention. This study was partially supported by grants from ‘‘Associazione Italiana per la Ricerca sul Cancro’’, and by the Environmental Cancer Risk, Nutrition and Individual Susceptibility European Union Network of Excellence (ECNIS, Contract No. FOOD-CT-2005- 513943) and a grant from NIH 5P20CA132385-03.

The ability to non-invasively monitor DNA oxidation and its repair has significant utility in large-scale, population-based studies. Such studies could include assessments of the efficacy of antioxidant intervention strategies, pathological roles of DNA oxidation in various disease states and population or inter-individual differences in antioxidant defence and DNA repair. The analysis of urine, or indeed any extracellular matrix, for 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), using chromatographic or immunoassay procedures, is by far the most popular method to non-invasively assess oxidative insult to the genome. The actual biological significance of the presence of extracellular 8-oxodG is still a subject for debate however. Studies are slowly ruling out confounding factors such as diet and cell turnover, which would leave endogenous processes, notably repair, as the sole source of extracellular 8-oxodG. The method described herein exploits the non-invasive properties of urine sampling, coupled with efficient extraction of 8-oxodG by a validated solid-phase extraction procedure. Subsequent analysis by liquid chromatography-tandem mass spectrometry has the advantages of sensitivity, internal standardisation and robust peak identification.

Cyclophosphamide and other bifunctional alkylating agents are potent animal teratogens inducing a variety of malformations. Although cyclophosphamide-induced DNA damage is implicated as a primary mechanism underlying the teratogenesis initiated by cyclophosphamide, additional insights into the complex nature of the teratogenic process have been hampered by the inability to analyze the primary teratogenic lesions, i.e., cyclophosphamide-DNA adducts. Using tandem mass spectrometry, we show that the monofunctional adduct N-(2-chloroethyl)-N-[2-(7-guaninyl)ethyl]amine (NOR-G) and bifunctional adduct N,N-bis[2-(7-guaninyl)ethyl]amine (G-NOR-G) can be detected in the DNA of organogenesis-stage rat embryos after an in vitro exposure to an embryotoxic concentration of activated cyclophosphamide, i.e., 4-hydroperoxycyclophosphamide.