Genetic and epigenetic regulation of AHR gene expression in MCF-7 breast cancer cells: role of the proximal promoter GC-rich region.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, contributes to carcinogenesis through its role in the regulation of cytochrome P450 1 (CYP1)-catalyzed metabolism of carcinogens. Here, we investigated genetic and epigenetic mechanisms that affect AhR expression. Analyses of the human AHR proximal promoter in MCF-7 human breast cancer cells using luciferase assays and electrophoretic mobility shift assays revealed multiple specificity protein (Sp) 1 binding sequences that are transcriptional activators in vitro.

Cytochrome P450-mediated metabolism and DNA binding of 2-amino-1,7-dimethylimidazo[4,5-g]quinoxaline and its carcinogenic isomer 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in mice.

2-Amino-1,7-dimethylimidazo[4,5-g]quinoxaline (MeIgQx) is a recently discovered heterocyclic aromatic amine (HAA) that is formed during the cooking of meats. MeIgQx is an isomer of 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), a rodent carcinogen and possible human carcinogen that also occurs in cooked meats. MeIgQx is a bacterial mutagen, but knowledge about its metabolism and carcinogenic potential is lacking.

DNA adduct formation of 4-aminobiphenyl and heterocyclic aromatic amines in human hepatocytes.

DNA adduct formation of the aromatic amine, 4-aminobiphenyl (4-ABP), a known human carcinogen present in tobacco smoke, and the heterocyclic aromatic amines (HAAs), 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), potential human carcinogens, which are also present in tobacco smoke or formed during the high-temperature cooking of meats, was investigated in freshly cultured human hepatocytes. The carcinogens (10 μM) were incubated with hepatocytes derived from eight different donors for time periods up to 24 h. The DNA adducts were quantified by liquid chromatography-electrospray ionization mass spectrometry with a linear quadrupole ion trap mass spectrometer.

Identification of carcinogen DNA adducts in human saliva by linear quadrupole ion trap/multistage tandem mass spectrometry.

DNA adducts of carcinogens derived from tobacco smoke and cooked meat were identified by liquid chromatography-electrospray ionization/multistage 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]pyridine (PhIP), 2-amino-9H-pyrido[2,3-b]indole (AalphaC), 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (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.

Biomonitoring of carcinogenic heterocyclic aromatic amines in hair: a validation study.

A facile method was established to measure heterocyclic aromatic amines (HAAs) accumulated in human hair and rodent fur. The samples were digested by base hydrolysis, and the liberated HAAs were isolated by tandem solvent/solid-phase extraction. Quantification was done by liquid chromatography/tandem mass spectrometry, using a triple stage quadrupole mass spectrometer in the selected reaction monitoring mode.

Investigations of the posttranslational mechanism of arsenite-mediated downregulation of human cytochrome P4501A1 levels: the role of heme oxygenase-1.

Arsenite, an environmental cocontaminant of polycyclic aromatic hydrocarbons (PAHs), diminishes the PAH-mediated upregulation of human CYP1A1, the enzyme that bioactivates PAHs to carcinogenic metabolites. Mechanistically, while transcriptional downregulation contributes to these effects, a role for posttranslational regulation has been implicated but not proven. We hypothesize that arsenite induces heme oxygenase-1 (HO-1), which catabolizes CYP1A1 heme or cellular heme pools, thereby downregulating CYP1A1.

Screening for DNA adducts by data-dependent constant neutral loss-triple stage mass spectrometry with a linear quadrupole ion trap mass spectrometer.

A two-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-MS3). The loss of the deoxyribose (dR) from the protonated DNA adducts ([M + H - 116]+) in the MS/MS scan mode triggered the acquisition of MS3 product ion spectra of the aglycone adducts [BH2]+. 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-MS3 scan mode.

Systems based mapping demonstrates that recovery from alkylation damage requires DNA repair, RNA processing, and translation associated networks.

The identification of cellular responses to damage can promote mechanistic insight into stress signalling. We have screened a library of 3968 Escherichia coli gene-deletion mutants to identify 99 gene products that modulate the toxicity of the alkylating agent methyl methanesulfonate (MMS). We have developed an ontology mapping approach to identify functional categories over-represented with MMS-toxicity modulating proteins and demonstrate that, in addition to DNA re-synthesis (replication, recombination, and repair), proteins involved in mRNA processing and translation influence viability after MMS damage.

Mechanisms of arsenite-mediated decreases in benzo[k]fluoranthene-induced human cytochrome P4501A1 levels in HepG2 cells.

Polycyclic aromatic hydrocarbons (PAHs) and heavy metals are often environmental cocontaminants that could interact to alter PAH carcinogenicity. The heavy metal, arsenite, and the PAH, benzo[k]fluoranthene, were used as prototypes to investigate, in human HepG2 cells, mechanisms whereby the bioactivation of benzo[k]fluoranthene by human CYP1A1 could be diminished by arsenite-mediated decreases in CYP1A1 induction by benzo[k]fluoranthene. To determine whether arsenite down-regulates CYP1A1 transcription, quantitative real-time reverse transcriptase-polymerase chain reaction assays and luciferase reporter gene expression assays were used with HepG2 cells treated with benzo[k]fluoranthene and arsenite, separately and as a mixture.