Endemic (Balkan) nephropathy (EN),environmental mutagen ͉ p53 mutation ͉ urothelial cancer ͉ DNA adduct

No abstract

An accurate and sensitive liquid chromatography-electrospray ionization/multi-stage mass spectrometry (LC-ESI/MS/MS n ) technique has been developed for the characterization and quantification of 2′-deoxyguanosine (dG) adducts of the dietary mutagen, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP). PhIP is an animal and potential human carcinogen that occurs in grilled meats. Following enzymatic digestion and adduct enrichment by solid-phase extraction (SPE), PhIP-DNA adducts were analyzed by MS/MS and MS n scan modes on a 2-D linear quadrupole ion trap mass spectrometer (QIT/MS). The major DNA adduct, N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (dG-C8-PhIP), was detected in calf thymus (CT) DNA modified in vitro with a bioactivated form of PhIP and in the colon and liver of rats given PhIP as part of the diet. The lower limit of detection (LOD) was 1 adduct per 10 8 DNA bases, and the limit of quantification (LOQ) was 3 adducts per 10 8 DNA bases in both MS/MS and MS 3 scan modes, using 27 μg of DNA for analysis. Measurements were based on isotope dilution with the internal standard, N-(deoxyguanosin-8-yl)-2-amino-1-(trideutero)methyl-6-phenylimidazo [4,5-b] pyridine (dG-C8-[ 2 H 3 C]-PhIP). The selected reaction monitoring (SRM) scan mode in MS/MS was employed to monitor the loss of deoxyribose (dR) from the protonated molecules of the adducts ([M + H -116] + ). The consecutive reaction monitoring (CRM) scan modes in MS 3 and MS 4 were used to measure and further characterize product ions of the aglycone ion (BH 2 + ) (Guanyl-PhIP). The MS 3 scan mode was effective in eliminating isobaric interferences observed in the MS/MS scan mode and resulted in an improved signal-to-noise (S/N) ratio. Moreover, the product ion spectra obtained by the MS n scan modes provided rich structural information about the adduct and were used to corroborate the identity of dG-C8-PhIP. In addition, an isomeric dG-PhIP adduct was detected in vivo. This LCESI/MS/MS n method is the first reported application on the use of the MS 3 scan mode for the analysis of DNA adducts in vivo.

A 2-dimensional linear quadrupole ion trap mass spectrometer (LIT/MS) was employed to simultaneously screen for DNA adducts of environmental, dietary, and endogenous genotoxicants, by data-dependent constant neutral loss scanning followed by triple-stage mass spectrometry (CNL-MS 3 ). The loss of the deoxyribose (dR) from the protonated DNA adducts ([M+H-116] + ) in the MS/ MS scan mode triggered the acquisition of MS 3 product ion spectra of the aglycone adducts [BH 2 + ]. Five DNA adducts of the tobacco carcinogen 4-aminobiphenyl (4-ABP) were detected in human hepatocytes treated with 4-ABP, and three DNA adducts of the cooked-meat carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) were identified in the livers of rats exposed to MeIQx, by the CNL-MS 3 scan mode. Buccal-cell DNA from tobacco smokers was screened for DNA adducts of various classes of carcinogens in tobacco smoke including 4-ABP, 2-amino-9H-pyrido [2,3-b]indole (AαC), and benzo[a]pyrene (BaP); the cooked-meat carcinogens MeIQx, AαC, and 2-amino-1-methyl-6-phenylmidazo[4,5-b]pyridine (PhIP); and the lipid peroxidation products acrolein (AC) and trans-4-hydroxynonenal (HNE). The CNL-MS 3 scanning technique can be used to simultaneously screen for multiple DNA adducts derived from different classes of carcinogens, at levels of adduct modification approaching 1 adduct per 10 8 unmodified DNA bases, when 10 μg of DNA are employed for the assay.

DNA adducts of carcinogens derived from tobacco smoke and cooked meat were identified, by liquid chromatography-electrospray ionization/multi-stage tandem mass spectrometry (LC-ESI/ MS/MS n ), in saliva samples from 37 human volunteers on unrestricted diets. The N-(deoxyguanosin-8-yl) (dG-C8) adducts of the heterocyclic aromatic amines 2-amino-1-methyl-6-phenylimidazo [4,5-b] (MeIQx); and the aromatic amine, 4-aminobiphenyl (4-ABP) were characterized and quantified, by LC-ESI/MS/MS n , employing consecutive reaction monitoring at the MS 3 scan stage mode with a linear quadrupole ion trap (LIT) mass spectrometer (MS). DNA adducts of PhIP were found most frequently: dG-C8-PhIP was detected in saliva samples from 13 of 29 ever-smokers and in saliva samples from 2 of 8 never-smokers. dG-C8-AαC and dG-C8-MeIQx were identified solely in saliva samples of 3 current smokers, and dG-C8-4-ABP was detected in saliva from 2 current-smokers. The levels of these different adducts ranged from 1 to 9 adducts per 10 8 DNA bases. These findings demonstrate that PhIP is a significant DNA-damaging agent in humans. Saliva appears to be a promising biological fluid in which to assay DNA adducts of tobacco and dietary carcinogens, by selective LIT MS techniques.

AIMSThis study aimed to assess changes in the plasma concentrationss of 4β-hydroxycholesterol (4βHC) against intravenous (i.v.) and oral midazolam (MDZ) pharmacokinetics (PK) after administration of a potent CYP3A inhibitor [ketoconazole (KETO)] and inducer [rifampicin (RIF)]. METHODSThirty-two healthy subjects (HS) were allocated into three groups of 12 each in KETO and RIF and 10 in a placebo group (PLB). All HS were randomized to receive oral and i.v. MDZ on day 1 or 2 and on day 15 or 16 after receiving RIF (600 mg once daily), KETO (400 mg once daily) or PLB for 2 weeks. Subjects were followed until day 30. The effect of treatments on 4βHC was assessed by analyzing % change from baseline using a linear spline mixed effects model. RESULTSCompared with PLB, KETO decreased 4βHC mean values up to 13% (P = 0.003) and RIF increased 4βHC mean values up to 220% (P < 0.001). Within 14 days of stopping KETO and RIF, 4βHC had either returned to baseline (KETO) or was still returning to baseline (RIF). Compared with baseline, mean oral MDZ AUC increased by 11-fold (90% CI ranging from 9-fold to 13-fold increase) and decreased by 92% (90% CI ranging from 90% to 95% decrease) after KETO and RIF, respectively. Similar trends were observed for 6β-hydroxycortisol : cortisol (6βHCL : CL) urinary ratios. CONCLUSIONSChanges in plasma 4βHC can be utilized as a surrogate for MDZ PK after multiple doses of potent CYP3A inducers. There is a more limited dynamic range for 4βHC for assessment of potential CYP3A inhibitors. 4βHC is a valuable tool for the assessment of potential CYP3A inducers in early drug development. WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT• Endogenous plasma 4β-hydroxycholesterol (4βHC) concentrations have been used as a putative CYP3A activity marker. However, placebo controlled studies have not been conducted to assess the dynamic range of potent CYP3A inhibitors and inducers. WHAT THIS STUDY ADDS• This placebo-controlled study assessed the dynamic range of plasma 4βHC concentrations after administration of ketoconazole and rifampicin (for 14 days). A comparison is made with other CYP3A trait measures such as midazolam pharmacokinetics and putative endogenous CYP3A biomarkers. The time course of CYP3A activity before and after administration of CYP3A modulators is also examined. This study validates 4βHC against midazolam pharmacokinetics and also expands the utility of 4βHC to assess CYP3A activity after stopping treatment with CYP3A inducers or inhibitors.• Results from this study show utility of 4βHC to defer or avoid midazolam-based studies, saving cost and avoiding exposing healthy subjects to study medications.

A previously unknown isomer of the carcinogenic heterocyclic aromatic amine (HAA) 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx) was recently discovered in urine of meat-eaters and subsequently detected in cooked ground beef [Holland, R.D. et al. (2004) Chem. Res. Toxicol. 17, 1121 -1136. In this current investigation, the identity of the analyte was determined through comparison of its chromatographic t R by HPLC, and through UV and mass spectral comparisons to the synthesized isomers of 8-MeIQx. Angular tricyclic isomers of 8-MeIQx were excluded as potential structures of the newly discovered HAA, based upon dissimilar t R and product ion mass spectral data. The linear tricyclic isomers 2-amino-1,6-dimethylimidazo[4,5-g]quinoxaline (6-MeIgQx) and 2-amino-1,7-dimethylimidazo[4,5-g]quinoxaline (7-MeIgQx) were postulated as plausible structures. Both compounds were synthesized from 4-fluoro-5-nitro-benzene-1,2-diamine in five steps. The structure of the analyte was proven to be 7-MeIgQx, based upon co-injection of the compound with the synthetic isomers, and corroborated by comparisons of the UV and mass spectral data of the analyte and MeIgQx isomers. 7-MeIgQx induced 348 revertants/μg in the S. typhimurium tester strain YG1024, when liver S-9 homogenate of rats pretreated with polychlorinated biphenyls (PCBs) was used for bioactivation. This newly discovered 7-MeIgQx molecule is one of the most abundant HAAs formed in cooked ground beef patties and pan-fried scrapings. The human health risk of 7-MeIgQx requires investigation.

Abstract1,N 2 -Etheno(ε)guanine (ε) is formed in DNA as a result of exposure to certain vinyl monomers (e.g. vinyl chloride) or from lipid peroxidation. This lesion has been shown to be mutagenic in bacteria and mammalian cells.