Cells deficient in the base excision repair protein, DNA polymerase beta, are hypersensitive to oxaliplatin chemotherapy.

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.

First-trimester increase in oxidative stress and risk of small-for-gestational-age fetus.

OBJECTIVE: Investigation of increased oxidative stress in early pregnancy and association with an increased risk of small-for-gestational-age (SGA) fetus. DESIGN: Longitudinal case-control study. SETTING: University Hospitals of Leicester NHS Trust, Leicester, UK. POPULATION: Low-risk pregnant women with no current or pre-existing medical illness were recruited at a large teaching hospital from 2004 to 2006. METHODS: Recruitment performed at the time of the dating ultrasound scan (12+/-2 weeks of gestation). Spot urine samples collected at 12+/-2 and 28+/-2 weeks of gestation were analysed for 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) by liquid chromatography with tandem mass spectrometry). SGA was defined as birthweight <10th centile based on customised centile calculator (www.gestation.net). This identified the cases (n=55), whereas controls (n=55) were mothers whose babies were appropriate for gestational age (AGA, birthweight 10th-90th centile). Statistical analysis was performed using GraphPad Prism v.5. The relationship between maternal urinary 8-oxodG at different gestations and customised SGA was investigated by nonparametric tests. MAIN OUTCOME MEASURES: Customised SGA and AGA pregnancies. RESULTS: Urinary 8-oxodG concentrations were significantly increased in pregnancies with subsequent SGA compared with concentrations in normal pregnancies; 12 weeks: 2.8 (interquartile range [IQR] 1.96-3.67) versus 2.2 (IQR 1.26-3.28) pmol 8-oxodG/micromol creatinine (P=0.0007); 28 weeks: 2.21 (IQR 1.67-3.14) versus 1.68 (IQR 1.16-2.82) pmol 8-oxodG/micromol creatinine (P<0.0002). Concentrations decreased significantly between week 12 and 28 (P=0.04 and P=0.02 for controls and cases). CONCLUSIONS: In this study, urinary 8-oxodG at 12 and 28 weeks were elevated in SGA compared with AGA pregnancies. This may reflect early placental changes predating clinical features of SGA.

Preclinical toxicokinetic evaluation of phortress [2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole lysylamide dihydrochloride] in two rodent species.

BACKGROUND AND AIMS: The 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole prodrug Phortress exerts potent and selective antitumour activity in vitro and in vivo. Preclinical toxicokinetic studies in 2 rodent species were undertaken to determine Phortress' maximum tolerated dose and advise a safe starting dose for clinical evaluation. METHODS: Plasma pharmacokinetic parameters were determined by high-performance liquid chromatography and fluorescence detection following Phortress administration to mice (10 mg/kg, intravenously on days 1 and 8). Phortress (20 mg/kg, on days 1 and 8) was administered to CYP1A1/betaGAL reporter mice; tissues were examined macro- and microscopically. Toxicological and pharmacodynamic endpoints were examined in organs of rodents receiving Phortress (10 mg/kg or 20 mg/kg, on days 1 and 8). CYP1A1 expression and Phortress-derived DNA adducts were determined in lungs and livers (on days 11 and 36). RESULTS: No accumulation of Phortress was detected in murine plasma. beta-Galactosidase activity inferred Phortress-derived induction of cyp1a1 transcription in the livers of transgenic mice; no total body weight loss was encountered in these animals. However, a fall in lung:body weight and kidney:body weight ratios, raised serum alkaline phosphatase levels and hepatic histopathological disturbances in animals receiving 20 mg/kg Phortress indicate organ sites of potential toxicity. CYP1A1 protein was induced transiently in the lungs of both species and in the livers of rats. Elimination of hepatic DNA adducts and rat pulmonary adducts was evident; however, murine pulmonary adducts persisted. CONCLUSION: Rodent preclinical toxicology established that mice represent the more sensitive rodent species, resolving a maximum tolerated dose of 10 mg/kg Phortress.

Styrene-oxide N-terminal valine haemoglobin adducts as biomarkers of occupational exposure to styrene.

Styrene is widely used in the production of various plastics, synthetic rubber and resins. Occupational exposure occurs mainly via inhalation and relatively high exposure occurs due to its use in manual application techniques. The aim of this study was to evaluate if SO-Hb adducts are a suitable biomarker for assessing occupational exposure to styrene. Seventy-five reinforced plastic workers and 77 control subjects were studied. In the selected population the main urinary styrene metabolites and the styrene oxide N-terminal valine (SO-Hb) adducts in human globin were quantified. The levels of SO-Hb adducts were significantly higher (p<0.01) in the exposed subjects (5.98pmol/g globin) when compared with controls (2.59pmol/g globin) and a significant difference was found in levels of SO-Hb adducts between non-smokers and smokers among the control group. From our data we conclude that SO-Hb adduct measurement is a sensitive and specific means of assessing exposure to styrene at the occupational and environmental level.

Styrene-oxide N-terminal valine haemoglobin adducts in reinforced plastic workers: possible influence of genetic polymorphism of drug-metabolising enzymes.

Styrene is one of the most important organic chemicals used worldwide. In humans, styrene metabolism involves oxidation by cytochrome P450 monooxygenases (CYPs) to styrene-7,8-oxide, an epoxide thought to be responsible for the genotoxic effects of styrene exposure, and detoxification by means of epoxide hydrolase (mEH) and glutathione S-transferases (GSTs). The objective of this study was to investigate if genetic polymorphisms of metabolic enzymes modulate the level of urinary styrene metabolites and styrene oxide adducts with N-terminal valine of human globin (SO-Hb) in 75 workers occupationally exposed to styrene and 77 unexposed controls. The mean air concentration of styrene in the breathing zone of workers (30.4ppm) was higher than the threshold limit value of 20ppm recommended by the American Conference of Governmental Industrial Hygienists (ACGIH), and the biological exposure index adopted by the ACGIH for exposure to styrene prior to the next shift (MA+PGA=400mg/g creatinine) was exceeded, indicating that styrene exposure for this group of workers was higher than recommended. A highly significant correlation was observed between styrene concentration in the breathing zone and the MA+PGA in urine of workers (r=0.85, P<0.001). The levels of SO-Hb adducts in exposed workers were significantly increased as compared with controls, although no difference was observed between subjects stratified as high and medium exposure categories based on MA+PGA excretion. Regarding the effect of the genetic polymorphisms we found that the level of SO-Hb adducts might be modulated by the predicted mEH enzymatic activity in the exposed workers. From our data we conclude that SO-Hb adduct measurement is a complementary method to MA+PG measurement for assessing exposure to styrene at occupational and environmental levels, which reflects a more extensive exposure period.

In vitro genotoxicity of PAH mixtures and organic extract from urban air particles part I: acellular assay.

Acellular assay of calf thymus DNA+/-rat liver microsomal S9 fraction coupled with (32)P-postlabelling was used to study the genotoxic potential of organic compounds bound onto PM10 particles collected in three European cities-Prague (CZ), Kosice (SK) and Sofia (BG) during summer and winter periods. B[a]P alone induced DNA adduct levels ranging from 4.8 to 768 adducts/10(8) nucleotides in the concentration dependent manner. However, a mixture of 8 c-PAHs with equimolar doses of B[a]P induced 3.7-757 adducts/10(8) nucleotides, thus suggesting the inhibition of DNA adduct forming activity by interaction among various PAHs. Comparison of DNA adduct levels induced by various EOMs indicates higher variability among seasons than among localities. DNA adduct levels for Prague collection site varied from 19 to 166 adducts/10(8) nucleotides, for Kosice from 22 to 85 and for Sofia from 6 to 144 adducts/10(8) nucleotides. Bioactivation with S9 microsomal fraction caused 2- to 7-fold increase in DNA adduct levels compared to -S9 samples, suggesting a crucial role of indirectly acting genotoxic EOM components, such as PAHs. We have demonstrated for the first time a significant positive correlation between B[a]P content in EOMs and total DNA adduct levels detected in the EOM treated samples (R=0.83; p=0.04). These results suggest that B[a]P content in EOM is an important factor for the total genotoxic potential of EOM and/or B[a]P is a good indicator of the presence of other genotoxic compounds causing DNA adducts. Even stronger correlation between the content of genotoxic compounds in EOMs and total DNA adduct levels detected (R=0.94; p=0.005) was found when eight c-PAHs were taken into the consideration. Our findings support a hypothesis that a relatively limited number of EOM components is responsible for a major part of its genotoxicity detectable as DNA adducts by (32)P-postlabelling.

In vitro genotoxicity of PAH mixtures and organic extract from urban air particles part II: human cell lines.

Principal aims of this study were at first, to find a relevant human derived cell line to investigate the genotoxic potential of PAH-containing complex mixtures and second, to use this cell system for the analysis of DNA adduct forming activity of organic compounds bound onto PM10 particles. Particles were collected by high volume air samplers during summer and winter periods in three European cities (Prague, Kosice, and Sofia), representing different levels of air pollution. The genotoxic potential of extractable organic matter (EOM) was compared with the genotoxic potential of individual carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) as well as their artificial mixtures. Metabolically competent human hepatoma HepG2 cells, confluent cultures of human diploid lung fibroblasts (HEL), and the human monocytic leukemia cell line THP-1 were used as models. DNA adducts were analyzed by (32)P-postlabeling. The total DNA adduct levels induced in HepG2 cells after exposure to EOMs were higher than in HEL cells treated under the same conditions (15-190 versus 2-15adducts/10(8) nucleotides, in HepG2 and HEL cells, respectively). THP-1 cells exhibited the lowest DNA adduct forming activity induced by EOMs (1.5-3.7adducts/10(8) nucleotides). A direct correlation between total DNA adduct levels and c-PAH content in EOM was found for all EOMs in HepG2 cells incubated with 50microg EOM/ml (R=0.88; p=0.0192). This correlation was even slightly stronger when B[a]P content in EOMs and B[a]P-like adduct spots were analyzed (R=0.90; p=0.016). As THP-1 cells possess a limited metabolic capacity for most c-PAHs to form DNA reactive intermediates and are also more susceptible to toxic effects of PAHs and various EOM components, this cell line seemed to be an inappropriate system for genotoxicity studies of PAH-containing complex mixtures. The seasonal variability of genotoxic potential of extracts was stronger than variability among the three localities studied. In HepG2 cells, the highest DNA adduct levels were induced by EOM collected in Prague in the winter period, followed by Sofia and Kosice. However, in the summer sampling period, the order was quite opposite: Kosice>Sofia>Prague. When the EOM content per m(3) of air was taken into consideration in order to compare real exposures of humans to genotoxic compounds in all three localities, extracts from respirable dust particles collected in Sofia exhibited the highest genotoxicity regardless of the sampling period. The results indicate that most of DNA adducts detected in cells incubated with EOMs have their origin in low concentrations of c-PAHs representing 0.03-0.17% of EOM total mass. Finally, our results suggest that HepG2 cells have a metabolic capacity for PAHs similar to human hepatocytes and represent therefore the best in vitro model for investigating the genotoxic potential of complex mixtures containing PAHs among the three cell lines tested in this study.

Cyclooxygenase-2 expression and oxidative DNA adducts in murine intestinal adenomas: modification by dietary curcumin and implications for clinical trials.

The natural polphenol, curcumin, retards the growth of intestinal adenomas in the Apc(Min+) mouse model of human familial adenomatous polyposis. In other preclinical models, curcumin downregulates the transcription of the enzyme cyclooxygenase-2 (COX-2) and decreases levels of two oxidative DNA adducts, the pyrimidopurinone adduct of deoxyguanosine (M1dG) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG). We have studied COX-2 protein expression and oxidative DNA adduct levels in intestinal adenoma tissue from Apc(Min+) mice to try and differentiate between curcumin's direct pharmacodynamic effects and indirect effects via its inhibition of adenoma growth. Mice received dietary curcumin (0.2%) for 4 or 14 weeks. COX-2 protein, M1dG and 8-oxo-dG levels were measured by Western blot, immunochemical assay and liquid chromatography-mass spectrometry, respectively. In control Apc(Min+) mice, the levels of all three indices measured in adenoma tissue were significantly higher than levels in normal mucosa. Lifetime administration of curcumin reduced COX-2 expression by 66% (P = 0.01), 8-oxo-dG levels by 24% (P < 0.05) and M1dG levels by 39% (P < 0.005). Short-term feeding did not affect total adenoma number or COX-2 expression, but decreased M1dG levels by 43% (P < 0.01). COX-2 protein levels related to adenoma size. These results demonstrate the utility of measuring these oxidative DNA adduct levels to show direct antioxidant effects of dietary curcumin. The effects of long-term dietary curcumin on COX-2 protein levels appear to reflect retardation of adenoma development.

DNA adducts: mass spectrometry methods and future prospects.

Detection of DNA adducts is widely used for the monitoring of exposure to genotoxic carcinogens. Knowledge of the nature and amounts of DNA adducts formed in vivo also gives valuable information regarding the mutational effects that may result from particular exposures. The power of mass spectrometry (MS) to achieve qualitative and quantitative analyses of human DNA adducts has increased greatly in recent years with the development of improved chromatographic interfaces and ionisation sources. Adducts have been detected on nucleic acid bases, 2'-deoxynucleosides or 2'-deoxynucleotides, with LC-MS/MS being the favoured technique for many of these analyses. Our current applications of this technique include the determination of N7-(2-carbamoyl-2-hydroxyethyl)-guanine, which was postulated to be found as a DNA repair product in urine following exposure to acrylamide, and of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydro-2'-deoxyadenosine, as markers of oxidative damage in human lymphocyte DNA. Higher sensitivity (with a detection limit of 1-10 adducts/10(12) nucleotides) may be achieved by the use of accelerator mass spectrometry (AMS), although this requires the presence of certain isotopes, such as [(14)C], in the material being analysed. In order to make this technique more amenable for studies of human exposure to environmental carcinogens, new postlabelling techniques, incorporating [(14)C] into specific DNA adducts after formation, are being developed. It is expected that combining the use of advanced MS techniques with existing (32)P-postlabelling and immunochemical methodologies will contribute greatly to the understanding of the burden of human exposure to environmental carcinogens.

Genotoxic damage in mine workers exposed to diesel exhaust, and the effects of glutathione transferase genotypes.

This study was performed in an Estonian shale-oil mine with the purpose to develop and apply a number of biomarkers for occupational diesel-exhaust exposure monitoring. Increased breathing-zone exposures to exhaust from operators of diesel-powered trucks in the mine was confirmed in the environmental monitoring part of the study, showing a 7.5-fold higher exposure to particle-associated 1-nitropyrene (1-NP) in 50 underground workers compared with 42 surface workers [P.T.J. Scheepers, D. Coggon, L.E. Knudsen, R. Anzion, H. Autrup, S. Bogovski, R.P. Bos, D. Dahmann, P. Farmer, E.A. Martin, V. Micka, V. Muzyka, H.-G. Neumann, J. Poole, A. Schmidt-Ott, F. Seiler, J. Volf, I. Zwirner-Baier, Biomarkers for occupational diesel exhaust exposure monitoring (BIOMODEM)-a study in underground mining, Toxicol. Lett. 134 (2002) 305-317; P.T.J. Scheepers, V. Micka, V. Muzyka, R. Anzion, D. Dahmann, J. Poole, R.P. Bos, Exposure to dust and particle-associated 1-nitropyrene of drivers of diesel-powered equipment in underground mining, Ann. Occp. Hyg. 47 (2003) 379-388]. Analysis of DNA damage by the Comet assay on frozen blood samples was performed on the total study group and showed significantly higher levels (p=0.003) in underground workers (smokers) driving diesel-powered excavation machines (median 155 on a scale from 0 to 400, among 47 persons), compared with surface workers who smoked (median of 90, among 46 persons). The level of DNA damage in underground smokers was significantly higher (p=0.04) than in non-smokers. Samples from 2 of the 3 sampling weeks had significantly lower DNA damage compared with the third week, probably due to timely processing and freezing. These samples also showed significant differences (p<0.001) between underground workers (median 145, among 41 persons) and surface workers (median 60, among 30 persons). An HPLC method was developed for the analysis of (32)P-postlabelled 1-NP-DNA-adducts, and was applied to a sub-sample of 20 workers. No significant differences between surface and underground workers were found in this sub-sample with respect to the minor, unidentified adducts that had similar chromatographic properties to 1-NP adducts, and smoking did not have any effect on adduct levels. No significant effects of the genotypes of GSTM1, GSTP1 and GSTT1 on DNA-adducts and on DNA damage as measured by the Comet assay were found in the total study group. The study confirms an increased level of DNA damage in workers exposed to exhaust from truck-driving in the mine. However, the results of the environmental and biological monitoring of 1-NP did not correlate, suggesting that inhalation exposure to diesel exhaust is not reflected by an increase in 1-NP-DNA-adduct levels and/or that factors other than occupational exposure to diesel exhaust are primary determinants of these DNA-adduct levels.

Human exposure and internal dose assessments of acrylamide in food.

This review provides a framework contributing to the risk assessment of acrylamide in food. It is based on the outcome of the ILSI Europe FOSIE process, a risk assessment framework for chemicals in foods and adds to the overall framework by focusing especially on exposure assessment and internal dose assessment of acrylamide in food. Since the finding that acrylamide is formed in food during heat processing and preparation of food, much effort has been (and still is being) put into understanding its mechanism of formation, on developing analytical methods and determination of levels in food, and on evaluation of its toxicity and potential toxicity and potential human health consequences. Although several exposure estimations have been proposed, a systematic review of key information relevant to exposure assessment is currently lacking. The European and North American branches of the International Life Sciences Institute, ILSI, discussed critical aspects of exposure assessment, parameters influencing the outcome of exposure assessment and summarised data relevant to the acrylamide exposure assessment to aid the risk characterisation process. This paper reviews the data on acrylamide levels in food including its formation and analytical methods, the determination of human consumption patterns, dietary intake of the general population, estimation of maximum intake levels and identification of groups of potentially high intakes. Possible options and consequences of mitigation efforts to reduce exposure are discussed. Furthermore the association of intake levels with biomarkers of exposure and internal dose, considering aspects of bioavailability, is reviewed, and a physiologically-based toxicokinetic (PBTK) model is described that provides a good description of the kinetics of acrylamide in the rat. Each of the sections concludes with a summary of remaining gaps and uncertainties.

Characterisation of metabolites of the putative cancer chemopreventive agent quercetin and their effect on cyclo-oxygenase activity.

Quercetin (3,5,7,3',4'-pentahydroxyflavone) is a flavone with putative ability to prevent cancer and cardiovascular diseases. Its metabolism was evaluated in rats and human. Rats received quercetin via the intravenous (i.v.) route and metabolites were isolated from the plasma, urine and bile. Analysis was by high-performance liquid chromatography and confirmation of species identity was achieved by mass spectrometry. Quercetin and isorhamnetin, the 3'-O-methyl analogue, were found in both the plasma and urine. In addition, several polar peaks were characterised as sulphated and glucuronidated conjugates of quercetin and isorhamnetin. Extension of the metabolism studies to a cancer patient who had received quercetin as an i.v. bolus showed that (Quercetin removed) isorhamnetin and quercetin 3'-O-sulphate were major plasma metabolites. As a catechol, quercetin can potentially be converted to a quinone and subsequently conjugated with glutathione (GSH). Oxidation of quercetin with mushroom tyrosinase in the presence of GSH furnished GSH conjugates of quercetin, two mono- and one bis-substituted conjugates. However, these species were not found in biomatrices in rats treated with quercetin. As cyclo-oxygenase-2 (COX-2) expression is mechanistically linked to carcinogenesis, we examined whether quercetin and its metabolites can inhibit COX-2 in a human colorectal cancer cell line (HCA-7). Isorhamnetin and its 4'-isomer tamarixetin were potent inhibitors, reflected in a 90% decrease in prostaglandin E-2 (PGE-2) levels, a marker of COX-2 activity. Quercetin was less effective, with a 50% decline. Quercetin 3- and 7-O-sulphate had no effect on PGE-2. The results indicate that quercetin may exert its pharmacological effects, at least in part, via its metabolites.

Detection of curcumin and its metabolites in hepatic tissue and portal blood of patients following oral administration.

Studies in vitro and in animal models of colorectal and hepatocellular cancers suggest that curcumin is an effective chemopreventive agent. In this pilot trial, we investigated whether oral administration of curcumin results in concentrations of the agent in normal and malignant human liver tissue, which are sufficient to elicit pharmacological activity. In total, 12 patients with hepatic metastases from colorectal cancer received 450-3600 mg of curcumin daily, for 1 week prior to surgery. Levels of curcumin and its metabolites were measured by HPLC in portal and peripheral blood, bile and liver tissue. Curcumin was poorly available, following oral administration, with low nanomolar levels of the parent compound and its glucuronide and sulphate conjugates found in the peripheral or portal circulation. While curcumin was not found in liver tissue, trace levels of products of its metabolic reduction were detected. In patients who had received curcumin, levels of malondialdehyde-DNA (M(1)G) adduct, which reflect oxidative DNA changes, were not decreased in post-treatment normal and malignant liver tissue when compared to pretreatment samples. The results suggest that doses of curcumin required to furnish hepatic levels sufficient to exert pharmacological activity are probably not feasible in humans.

Pharmacokinetics in mice and growth-inhibitory properties of the putative cancer chemopreventive agent resveratrol and the synthetic analogue trans 3,4,5,4'-tetramethoxystilbene.

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.

Antitumour 2-(4-aminophenyl)benzothiazoles generate DNA adducts in sensitive tumour cells in vitro and in vivo.

2-(4-Aminophenyl)benzothiazoles represent a potent and highly selective class of antitumour agent. In vitro, sensitive carcinoma cells deplete 2-(4-aminophenyl)benzothiazoles from nutrient media; cytochrome P450 1A1 activity, critical for execution of antitumour activity, and protein expression are powerfully induced. 2-(4-Amino-3-methylphenyl)benzothiazole-derived covalent binding to cytochrome P450 1A1 is reduced by glutathione, suggesting 1A1-dependent production of a reactive electrophilic species. In vitro, 2-(4-aminophenyl)benzothiazole-generated DNA adducts form in sensitive tumour cells only. At concentrations >100 nM, adducts were detected in DNA of MCF-7 cells treated with 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203). 5F 203 (1 microM) led to the formation of one major and a number of minor adducts. However, treatment of cells with 10 microM 5F 203 resulted in the emergence of a new dominant adduct. Adducts accumulated steadily within DNA of MCF-7 cells exposed to 1 microM 5F 203 between 2 and 24 h. Concentrations of the lysylamide prodrug of 5F 203 (Phortress) > or = 100 nM generated adducts in the DNA of sensitive MCF-7 and IGROV-1 ovarian cells. At 1 microM, one major Phortress-derived DNA adduct was detected in these two sensitive phenotypes; 10 microM Phortress led to the emergence of an additional major adduct detected in the DNA of MCF-7 cells. Inherently resistant MDA-MB-435 breast carcinoma cells incurred no DNA damage upon exposure to Phortress (< or = 10 microM, 24 h). In vivo, DNA adducts accumulated within sensitive ovarian IGROV-1 and breast MCF-7 xenografts 24 h after treatment of mice with Phortress (20 mg kg(-1)). Moreover, Phortress-derived DNA adduct generation distinguished sensitive MCF-7 tumours from inherently resistant MDA-MB-435 xenografts implanted in opposite flanks of the same mouse.

The cancer preventative agent resveratrol is converted to the anticancer agent piceatannol by the cytochrome P450 enzyme CYP1B1.

Resveratrol is a cancer preventative agent that is found in red wine. Piceatannol is a closely related stilbene that has antileukaemic activity and is also a tyrosine kinase inhibitor. Piceatannol differs from resveratrol by having an additional aromatic hydroxy group. The enzyme CYP1B1 is overexpressed in a wide variety of human tumours and catalyses aromatic hydroxylation reactions. We report here that the cancer preventative agent resveratrol undergoes metabolism by the cytochrome P450 enzyme CYP1B1 to give a metabolite which has been identified as the known antileukaemic agent piceatannol. The metabolite was identified by high performance liquid chromatography analysis using fluorescence detection and the identity of the metabolite was further confirmed by derivatisation followed by gas chromatography-mass spectrometry studies using authentic piceatannol for comparison. This observation provides a novel explanation for the cancer preventative properties of resveratrol. It demonstrates that a natural dietary cancer preventative agent can be converted to a compound with known anticancer activity by an enzyme that is found in human tumours. Importantly this result gives insight into the functional role of CYP1B1 and provides evidence for the concept that CYP1B1 in tumours may be functioning as a growth suppressor enzyme.

Evaluation of phosphodiesterase I-based protocols for the detection of multiply damaged sites in DNA: the detection of abasic, oxidative and alkylative tandem damage in DNA oligonucleotides.

It has been proposed that DNA multiply damaged sites (MDS), where more than one moiety in a local region ( approximately 1 helical turn, 10 bp) of the DNA is damaged, are lesions of enhanced biological significance. However, other than indirect measures, there are few analytical techniques that allow direct detection of MDS in DNA. In the present study we demonstrate the potential of protocols incorporating an exonucleolytic snake venom phosphodiesterase (SVPD) digestion stage to permit the direct detection of certain tandem damage, in which two lesions are immediately adjacent to each other on the same DNA strand. A series of prepared oligonucleotides containing either single or pairs of tetrahydrofuran moieties (F), thymine glycol lesions (T(g)) or methylphosphotriester adducts (Me-PTE) were digested with SVPD and the digests examined by either (32)P-end-labelling or electrospray mass spectrometry. The unambiguous observation of SVPD-resistant 'trimer' species in the digests of oligonucleotides containing adjacent F, T(g) and Me-PTE demonstrates that the SVPD digestion strategy is capable of allowing direct detection of certain tandem damage. Furthermore, in studies to determine the specificity of SVPD in dealing with pairs of lesions on the same strand, it was found mandatory to have the two lesions immediately adjacent to each other in order to generate the trimer species; pairs of lesions separated by as few as one or two normal nucleotides behave principally as single lesions towards SVPD.

Presence of benzo[a]pyrene diol epoxide adducts in target DNA leads to an increase in UV-induced DNA single strand breaks and supF gene mutations.

Exposure to DNA damaging agents and mutagens often occurs as combinations of agents, or as complex mixtures of chemicals. We found that plasmid DNA adducted with benzo[a]pyrene diol epoxide (BPDE) was more susceptible to UV-induced single strand breaks than was control DNA. To determine whether the increase in DNA damage also applied to mutagenic lesions, the supF gene forward mutation assay was used to compare mutations induced by BPDE alone, UVB, UVC, BPDE followed by UVB and BPDE followed by UVC. It was found that the mutation frequency for BPDE + UVB (1167 in 10(4) transformants) was higher than BPDE alone (12 in 10(4) transformants) or UVB alone (446 in 10(4) transformants), and the mutation frequency for BPDE + UVC (197 in 10(4) transformants) was higher than BPDE alone or UVC alone (26 in 10(4) transformants). For BPDE + UVB and BPDE + UVC there was a significant increase in plasmids with multiple mutations. Whilst these indicate error prone repair due to the single strand breaks, the different mutation frequencies in plasmids treated to give similar levels of strand breaks suggest other mechanisms for the mutations in plasmids with single mutation events. The spectrum of non-multiple mutations in the two combined treatments included both UV signature mutations (GC-->AT as the most common mutation) and BPDE signature mutations (GC-->TA and GC-->CG as the most common mutations). However, the increase in absolute mutation frequency of BPDE signature mutations between BPDE treatment and BPDE + UV treatment was greater than the increase in absolute mutation frequency of UV signature mutations, even though the level of BPDE adducts was identical in each case. These results suggest two possibilities: (i) the BPDE adducts are photoactivated to a more mutagenic lesion, or (ii) the presence of UV lesions lead to the BPDE adducts becoming more mutagenic.

Isolation and characterization of protoporphyrin glycoconjugates from rat harderian gland by HPLC, capillary electrophoresis and HPLC/electrospray ionization MS.

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 beta-xyloside with a smaller amount of protoporphyrin-1-O-acyl beta-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.

Methods of DNA adduct determination and their application to testing compounds for genotoxicity.

At the International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC (March 25-26, 1999), a working group considered the uses of DNA adduct determination methods for testing compounds for genotoxicity. When a drug or chemical displays an unusual or inconsistent combination of positive and negative results in in vitro and in vivo genotoxicity assays and/or in carcinogenicity experiments, investigations into whether or not DNA adducts are formed may be helpful in assessing whether or not the test compound is a genotoxin. DNA adduct determinations can be carried out using radiolabeled compounds and measuring radioactive decay (scintillation counting) or isotope ratios (accelerator mass spectrometry) in the isolated DNA. With unlabeled compounds adducts may be measured by (32)P-postlabeling analysis of the DNA, or by physicochemical methods including mass spectrometry, fluorescence spectroscopy, or electrochemical detection, or by immunochemical methods. Each of these approaches has different strengths and limitations, influenced by sensitivity, cost, time, and interpretation of results. The design of DNA binding studies needs to be on a case-by-case basis, depending on the compound's profile of activity. DNA purity becomes increasingly important the more sensitive, and less chemically specific, the assay. While there may be adduct levels at which there is no observable biological effect, there are at present insufficient data on which to set a threshold level for biological significance.