Benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are carcinogens, ... more Benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are carcinogens, which frequently occur in the human diet. Their metabolic activation to reactive species binding to DNA is mediated by cytochromes P450 (CYPs) 1A1 and 1A2. Thus, levels and activities of these CYPs are crucial for initiation of BaP- and PhPI-mediated carcinogenesis. Here, the effect of CYP1A1/2 induction due to their prototype flavonoid inducer, β-naphthoflavone (BNF), on BaP- and PhPI-derived DNA adduct formation in rats was examined. Male rats pretreated with BNF were treated with a single dose of either carcinogen by oral gavage. Nuclease P1 version of 32P-postlabeling assay and online column-switching liquid chromatography-electrospray ionization-tandem mass spectrometry were used to detect and quantify covalent DNA adducts formed by BaP and PhIP in-vivo, respectively. Expression of CYP1A1/2 enzymes was examined by Western blot. Enzymatic activities of CYP1A1/2 were assessed using the...
ABSTRACT The relative partial ionization cross sections for the fragment ions produced in direct ... more ABSTRACT The relative partial ionization cross sections for the fragment ions produced in direct and dissociative ionization of a N2O molecule are measured for impact of 10-25-keV electrons by using an electron-ion-coincidence technique with a linear time-of-flight spectrometer. The six ionic fragments of N2O (N2O+, NO+, N2+, O+, N+, and N2+ + O2+) are observed and identified. The impact energy dependence of the partial ionization cross sections for these ions is expressed relative to the cross section of N2O+ and is found to be nearly invariant. The relative ionic fractions for the produced ions of N2O are also obtained and compared with the earlier reported data available at lower energies of electron impact. It is found that the relative ionic fractions for singly charged fragments are almost energy independent. However, for the doubly charged fragment ions (N2+ + O2+), the present data are found to be higher by almost a factor of four compared to the relative ionic fraction reported earlier at a very low impact energy.
A significant proportion of human cancers overexpress DNA polymerase beta (Pol beta), the major D... more 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.
We describe a new experimental setup for studying the fragmentation dynamics of molecules induced... more We describe a new experimental setup for studying the fragmentation dynamics of molecules induced by the impact of keV electrons using the well-known technique of recoil ion momentum spectroscopy. The apparatus consists of mainly a time- and position-sensitive multi-hit particle detector for ion analysis and a channel electron multiplier detector for detecting the ejected electrons. Different components of the setup and the relevant electronics for data acquisition are described in detail with their working principles. In order to verify the reliable performance of the setup, we have recorded the collision-induced ionic spectra of the CO2 molecule by the impact of keV electrons. Information about the ion pairs of CO+:O+, C+:O+ and O+:O+ resulting from dissociative ionizing collisions of 20 and 26 keV electrons with a dilute gaseous target of CO2 molecules has been obtained. Under conditions of the present experiment, the momentum resolutions of the spectrometer for the combined momenta of CO+ and O+ ions in the direction of the time-of-flight axis and perpendicular to the direction of an electron beam are found to be 10.0 ± 0.2 and 15.0 ± 0.3 au, respectively.
Journal of Electron Spectroscopy and Related Phenomena, 2012
We present the measurements of total M-shell X-ray yields Y(E 0) of a pure thick Pt element produ... more We present the measurements of total M-shell X-ray yields Y(E 0) of a pure thick Pt element produced by electron impact in the energy range of E 0 = 10-25 keV. Measurements are performed by employing a Si-PIN photo diode X-ray detector as an energy-dispersive spectrometer (EDS). The experimental data for Y(E 0) are compared with the corresponding predictions from Monte Carlo (MC) calculations using the general purpose MC code PENELOPE. A good agreement is obtained between the experiment and the MC calculations for the variation of Y(E 0) with impact energy both in shape and in magnitude within the experimental uncertainty of about 12.5%.
Melorrheostosis is a rare osteosclerotic bone dysplasia of obscure etiology. The typical radiogra... more Melorrheostosis is a rare osteosclerotic bone dysplasia of obscure etiology. The typical radiographic features are flowing candle wax, sub-periosteal bone and streaky endosteal bone formation in diaphyseal and epiphyseal area with sclerotomal pattern mainly involving appendicular skeleton. It is rarely associated with nerve palsies. The authors report a case of melorrheostotic mass causing sciatic neuropraxia and to the best of their knowledge it is the first case reported in the English language literature.
The heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed... more 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)-2amino-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 columnswitching 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 µg 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 4 interfere with the mass spectrometric analysis, with sufficient sensitivity and precision to analyse DNA adducts in animals exposed to PhIP.
Antiangiogenic gene transfer has the potential to be more efficacious than protein-based therapie... more Antiangiogenic gene transfer has the potential to be more efficacious than protein-based therapies or pharmacotherapies for the control of solid tumor growth, invasion and metastasis. For a sustained antiangiogenic effect, a vector capable of long-term expression without vector-associated immunity or toxicity is advantageous. The present study evaluated the potential of a recombinant adeno-associated virus-2 (rAAV) encoding the human soluble FMS-like tyrosine kinase receptor 1 (sFlt-1), which functions by both sequestering vascular endothelial growth factor (VEGF) and forming inactive heterodimers with other membranespanning VEGF receptors, in vitro and in vivo. Results indicated significant growth inhibitory activity of the transgenic factor in a human umbilical vein endothelial cell proliferation assay in vitro and protection against the growth of an angiogenesis-dependent human ovarian cancer cell line, SKOV3.ip1, xenograft in vivo with increased disease-free survival. Stable expression of the secretory factor and transgene persistence were confirmed by immunohistochemistry and in situ hybridization analyses, respectively. Increased therapeutic effects on both the growth index of the implanted tumor cells and tumor-free survival also correlated with an increasing dose of the vector used. These studies indicate that rAAV-mediated sFlt-1 gene therapy may be a feasible approach for inhibiting tumor angiogenesis, particularly as an adjuvant/therapy.
The formation of DNA adducts by the covalent binding of genotoxic chemicals to DNA represents a v... more 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.
Biochemical and Biophysical Research Communications, 2013
ELISA is widely used for urinary 8-oxo-7,8-dihydro-2 0-deoxyguanosine (8-oxodG) analysis. It is t... more ELISA is widely used for urinary 8-oxo-7,8-dihydro-2 0-deoxyguanosine (8-oxodG) analysis. It is the method of choice for laboratories that lack specialized chromatographic instrumentation. It allows fast, high-throughput sample analysis without a need for extensive samples processing. However, a lack of agreement between ELISA and chromatographic methods confines its application to the assessment of relative urinary 8-oxodG levels. We investigated various ELISA modifications, seeking optimal conditions that would yield a good agreement between ELISA and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Purification of urine by solid phase extraction (SPE), then incubation with the anti-8-oxodG antibody at 4°C overnight and subsequent normalization of 8-oxodG levels per urinary creatinine resulted in a near-perfect correlation and agreement in mean levels between ELISA and HPLC-MS/MS (r = 0.917, p < 0.001; and paired t-test p = 0.803, respectively). Our data show that, after introduction of a simple modification, ELISA quantification urinary 8-oxodG substantially improves. Although more sample manipulation is required, the method retains its key advantages over chromatography (high-throughput analysis that does not require expensive instrumentation). This represents a significant advance for the ELISA, and encouraging its use in more studies adding to our knowledge of the role of this biomarker of oxidative stress in health and disease.
Aims: Urinary 8-oxo-7,8-dihydro-2¢-deoxyguanosine (8-oxodG) is a widely used biomarker of oxidati... more Aims: Urinary 8-oxo-7,8-dihydro-2¢-deoxyguanosine (8-oxodG) is a widely used biomarker of oxidative stress. However, variability between chromatographic and ELISA methods hampers interpretation of data, and this variability may increase should urine composition differ between individuals, leading to assay interference. Furthermore, optimal urine sampling conditions are not well defined. We performed inter-laboratory comparisons of 8-oxodG measurement between mass spectrometric-, electrochemical-and ELISA-based methods, using common within-technique calibrants to analyze 8-oxodG-spiked phosphate-buffered saline and urine samples. We also investigated human subject-and sample collection-related variables, as potential sources of variability. Results: Chromatographic assays showed high agreement across urines from different subjects, whereas ELISAs
Helicobacter pylori infection is associated with elevated gastric mucosal concentrations of the l... more Helicobacter pylori infection is associated with elevated gastric mucosal concentrations of the lipid peroxidation product malondialdehyde and reduced gastric juice vitamin C concentrations. Malondialdehyde can react with DNA bases to form the mutagenic adduct ...
Benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are carcinogens, ... more Benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are carcinogens, which frequently occur in the human diet. Their metabolic activation to reactive species binding to DNA is mediated by cytochromes P450 (CYPs) 1A1 and 1A2. Thus, levels and activities of these CYPs are crucial for initiation of BaP- and PhPI-mediated carcinogenesis. Here, the effect of CYP1A1/2 induction due to their prototype flavonoid inducer, β-naphthoflavone (BNF), on BaP- and PhPI-derived DNA adduct formation in rats was examined. Male rats pretreated with BNF were treated with a single dose of either carcinogen by oral gavage. Nuclease P1 version of 32P-postlabeling assay and online column-switching liquid chromatography-electrospray ionization-tandem mass spectrometry were used to detect and quantify covalent DNA adducts formed by BaP and PhIP in-vivo, respectively. Expression of CYP1A1/2 enzymes was examined by Western blot. Enzymatic activities of CYP1A1/2 were assessed using the...
ABSTRACT The relative partial ionization cross sections for the fragment ions produced in direct ... more ABSTRACT The relative partial ionization cross sections for the fragment ions produced in direct and dissociative ionization of a N2O molecule are measured for impact of 10-25-keV electrons by using an electron-ion-coincidence technique with a linear time-of-flight spectrometer. The six ionic fragments of N2O (N2O+, NO+, N2+, O+, N+, and N2+ + O2+) are observed and identified. The impact energy dependence of the partial ionization cross sections for these ions is expressed relative to the cross section of N2O+ and is found to be nearly invariant. The relative ionic fractions for the produced ions of N2O are also obtained and compared with the earlier reported data available at lower energies of electron impact. It is found that the relative ionic fractions for singly charged fragments are almost energy independent. However, for the doubly charged fragment ions (N2+ + O2+), the present data are found to be higher by almost a factor of four compared to the relative ionic fraction reported earlier at a very low impact energy.
A significant proportion of human cancers overexpress DNA polymerase beta (Pol beta), the major D... more 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.
We describe a new experimental setup for studying the fragmentation dynamics of molecules induced... more We describe a new experimental setup for studying the fragmentation dynamics of molecules induced by the impact of keV electrons using the well-known technique of recoil ion momentum spectroscopy. The apparatus consists of mainly a time- and position-sensitive multi-hit particle detector for ion analysis and a channel electron multiplier detector for detecting the ejected electrons. Different components of the setup and the relevant electronics for data acquisition are described in detail with their working principles. In order to verify the reliable performance of the setup, we have recorded the collision-induced ionic spectra of the CO2 molecule by the impact of keV electrons. Information about the ion pairs of CO+:O+, C+:O+ and O+:O+ resulting from dissociative ionizing collisions of 20 and 26 keV electrons with a dilute gaseous target of CO2 molecules has been obtained. Under conditions of the present experiment, the momentum resolutions of the spectrometer for the combined momenta of CO+ and O+ ions in the direction of the time-of-flight axis and perpendicular to the direction of an electron beam are found to be 10.0 ± 0.2 and 15.0 ± 0.3 au, respectively.
Journal of Electron Spectroscopy and Related Phenomena, 2012
We present the measurements of total M-shell X-ray yields Y(E 0) of a pure thick Pt element produ... more We present the measurements of total M-shell X-ray yields Y(E 0) of a pure thick Pt element produced by electron impact in the energy range of E 0 = 10-25 keV. Measurements are performed by employing a Si-PIN photo diode X-ray detector as an energy-dispersive spectrometer (EDS). The experimental data for Y(E 0) are compared with the corresponding predictions from Monte Carlo (MC) calculations using the general purpose MC code PENELOPE. A good agreement is obtained between the experiment and the MC calculations for the variation of Y(E 0) with impact energy both in shape and in magnitude within the experimental uncertainty of about 12.5%.
Melorrheostosis is a rare osteosclerotic bone dysplasia of obscure etiology. The typical radiogra... more Melorrheostosis is a rare osteosclerotic bone dysplasia of obscure etiology. The typical radiographic features are flowing candle wax, sub-periosteal bone and streaky endosteal bone formation in diaphyseal and epiphyseal area with sclerotomal pattern mainly involving appendicular skeleton. It is rarely associated with nerve palsies. The authors report a case of melorrheostotic mass causing sciatic neuropraxia and to the best of their knowledge it is the first case reported in the English language literature.
The heterocyclic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed... more 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)-2amino-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 columnswitching 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 µg 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 4 interfere with the mass spectrometric analysis, with sufficient sensitivity and precision to analyse DNA adducts in animals exposed to PhIP.
Antiangiogenic gene transfer has the potential to be more efficacious than protein-based therapie... more Antiangiogenic gene transfer has the potential to be more efficacious than protein-based therapies or pharmacotherapies for the control of solid tumor growth, invasion and metastasis. For a sustained antiangiogenic effect, a vector capable of long-term expression without vector-associated immunity or toxicity is advantageous. The present study evaluated the potential of a recombinant adeno-associated virus-2 (rAAV) encoding the human soluble FMS-like tyrosine kinase receptor 1 (sFlt-1), which functions by both sequestering vascular endothelial growth factor (VEGF) and forming inactive heterodimers with other membranespanning VEGF receptors, in vitro and in vivo. Results indicated significant growth inhibitory activity of the transgenic factor in a human umbilical vein endothelial cell proliferation assay in vitro and protection against the growth of an angiogenesis-dependent human ovarian cancer cell line, SKOV3.ip1, xenograft in vivo with increased disease-free survival. Stable expression of the secretory factor and transgene persistence were confirmed by immunohistochemistry and in situ hybridization analyses, respectively. Increased therapeutic effects on both the growth index of the implanted tumor cells and tumor-free survival also correlated with an increasing dose of the vector used. These studies indicate that rAAV-mediated sFlt-1 gene therapy may be a feasible approach for inhibiting tumor angiogenesis, particularly as an adjuvant/therapy.
The formation of DNA adducts by the covalent binding of genotoxic chemicals to DNA represents a v... more 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.
Biochemical and Biophysical Research Communications, 2013
ELISA is widely used for urinary 8-oxo-7,8-dihydro-2 0-deoxyguanosine (8-oxodG) analysis. It is t... more ELISA is widely used for urinary 8-oxo-7,8-dihydro-2 0-deoxyguanosine (8-oxodG) analysis. It is the method of choice for laboratories that lack specialized chromatographic instrumentation. It allows fast, high-throughput sample analysis without a need for extensive samples processing. However, a lack of agreement between ELISA and chromatographic methods confines its application to the assessment of relative urinary 8-oxodG levels. We investigated various ELISA modifications, seeking optimal conditions that would yield a good agreement between ELISA and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Purification of urine by solid phase extraction (SPE), then incubation with the anti-8-oxodG antibody at 4°C overnight and subsequent normalization of 8-oxodG levels per urinary creatinine resulted in a near-perfect correlation and agreement in mean levels between ELISA and HPLC-MS/MS (r = 0.917, p < 0.001; and paired t-test p = 0.803, respectively). Our data show that, after introduction of a simple modification, ELISA quantification urinary 8-oxodG substantially improves. Although more sample manipulation is required, the method retains its key advantages over chromatography (high-throughput analysis that does not require expensive instrumentation). This represents a significant advance for the ELISA, and encouraging its use in more studies adding to our knowledge of the role of this biomarker of oxidative stress in health and disease.
Aims: Urinary 8-oxo-7,8-dihydro-2¢-deoxyguanosine (8-oxodG) is a widely used biomarker of oxidati... more Aims: Urinary 8-oxo-7,8-dihydro-2¢-deoxyguanosine (8-oxodG) is a widely used biomarker of oxidative stress. However, variability between chromatographic and ELISA methods hampers interpretation of data, and this variability may increase should urine composition differ between individuals, leading to assay interference. Furthermore, optimal urine sampling conditions are not well defined. We performed inter-laboratory comparisons of 8-oxodG measurement between mass spectrometric-, electrochemical-and ELISA-based methods, using common within-technique calibrants to analyze 8-oxodG-spiked phosphate-buffered saline and urine samples. We also investigated human subject-and sample collection-related variables, as potential sources of variability. Results: Chromatographic assays showed high agreement across urines from different subjects, whereas ELISAs
Helicobacter pylori infection is associated with elevated gastric mucosal concentrations of the l... more Helicobacter pylori infection is associated with elevated gastric mucosal concentrations of the lipid peroxidation product malondialdehyde and reduced gastric juice vitamin C concentrations. Malondialdehyde can react with DNA bases to form the mutagenic adduct ...
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