Mass Spectrometric Analysis of the Active Site Tryptic Peptide of Recombinant O6-Methylguanine-DNA Methyltransferase Following Incubation with Human Colorectal DNA Reveals the Presence of an O6-Alkylguanine Adductome.

Human exposure to DNA alkylating agents is poorly characterized, partly because only a limited range of specific alkyl DNA adducts have been quantified. The human DNA repair protein, O6-methylguanine O6-methyltransferase (MGMT), irreversibly transfers the alkyl group from DNA O6-alkylguanines (O6-alkGs) to an acceptor cysteine, allowing the simultaneous detection of multiple O6-alkG modifications in DNA by mass spectrometric analysis of the MGMT active site peptide (ASP). Recombinant MGMT was incubated with oligodeoxyribonucleotides (ODNs) containing different O6-alkGs, Temozolomide-methylated calf thymus DNA (Me-CT-DNA), or human colorectal DNA of known O6-MethylG (O6-MeG) levels. It was digested with trypsin, and ASPs were detected and quantified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. ASPs containing S-methyl, S-ethyl, S-propyl, S-hydroxyethyl, S-carboxymethyl, S-benzyl, and S-pyridyloxobutyl cysteine groups were detected by incubating MGMT with ODNs containing the corresponding O6-alkGs. The LOQ of ASPs containing S-methylcysteine detected after MGMT incubation with Me-CT-DNA was <0.05 pmol O6-MeG per mg CT-DNA. Incubation of MGMT with human colorectal DNA produced ASPs containing S-methylcysteine at levels that correlated with those of O6-MeG determined previously by HPLC-radioimmunoassay (r2 = 0.74; p = 0.014). O6-CMG, a putative O6-hydroxyethylG adduct, and other potential unidentified MGMT substrates were also detected in human DNA samples. This novel approach to the identification and quantitation of O6-alkGs in human DNA has revealed the existence of a human DNA alkyl adductome that remains to be fully characterized. The methodology establishes a platform for characterizing the human DNA O6-alkG adductome and, given the mutagenic potential of O6-alkGs, can provide mechanistic information about cancer pathogenesis.

Atl1 regulates choice between global genome and transcription-coupled repair of O(6)-alkylguanines.

Nucleotide excision repair (NER) has long been known to remove DNA lesions induced by chemical carcinogens, and the molecular mechanism has been partially elucidated. Here we demonstrate that in Schizosaccharomyces pombe a DNA recognition protein, alkyltransferase-like 1 (Atl1), can play a pivotal role in selecting a specific NER pathway, depending on the nature of the DNA modification. The relative ease of dissociation of Atl1 from DNA containing small O(6)-alkylguanines allows accurate completion of global genome repair (GGR), whereas strong Atl1 binding to bulky O(6)-alkylguanines blocks GGR, stalls the transcription machinery, and diverts the damage to transcription-coupled repair. Our findings redraw the initial stages of the NER process in those organisms that express an alkyltransferase-like gene and raise the question of whether or not O(6)-alkylguanine lesions that are poor substrates for the alkyltransferase proteins in higher eukaryotes might, by analogy, signal such lesions for repair by NER.

The nitrosated bile acid DNA lesion O6-carboxymethylguanine is a substrate for the human DNA repair protein O6-methylguanine-DNA methyltransferase.

The consumption of red meat is a risk factor in human colorectal cancer (CRC). One hypothesis is that red meat facilitates the nitrosation of bile acid conjugates and amino acids, which rapidly convert to DNA-damaging carcinogens. Indeed, the toxic and mutagenic DNA adduct O(6)-carboxymethylguanine (O(6)-CMG) is frequently present in human DNA, increases in abundance in people with high levels of dietary red meat and may therefore be a causative factor in CRC. Previous reports suggested that O(6)-CMG is not a substrate for the human version of the DNA damage reversal protein O(6)-methylguanine-DNA methyltransferase (MGMT), which protects against the genotoxic effects of other O(6)-alkylguanine lesions by removing alkyl groups from the O(6)-position. We now show that synthetic oligodeoxyribonucleotides containing the known MGMT substrate O(6)-methylguanine (O(6)-MeG) or O(6)-CMG effectively inactivate MGMT in vitro (IC50 0.93 and 1.8 nM, respectively). Inactivation involves the removal of the O(6)-alkyl group and its transfer to the active-site cysteine residue of MGMT. O(6)-CMG is therefore an MGMT substrate, and hence MGMT is likely to be a protective factor in CRC under conditions where O(6)-CMG is a potential causative agent.

Working Conditions and Health Risk Assessment in Hair Salons.

Objective:The aim of this study was to assess the chemical and physical work conditions in hair salons and to analyze health risk of exposure to VOCs for the hairdressers of hair salons. Methods: This study was carried out at 4 selected hair salons close to universities in Surat Thani province, Thailand. VOCs were collected by area sampling using charcoal tubes (9 samples per salon). The air samples were analyzed using GC/FID. The noise levels, illumination, and temperature were measured by using sound level meter, lux meter, and WBGT, respectively. Results: Toluene, cyclohexanone, xylene, and hexane were the most frequently found across the hair salons. All of the VOCs concentration was highest in the mixing area. All of hair salons had cancer risk exceeding 1 × 10-6 and HI > 1.0, indicating that indoor air pollution may affect hairdresser's health. The average VOCs concentrations after installation of local exhaust ventilation and open the door for 30 minutes before closing the hair salon, was significantly lower than before and after installation of the local exhaust ventilation. The WBGT indoors varied within 22 to 28°C, sound pressure levels within 71 to 76 dBA, and illumination within 70 to 400 lux. The noise levels and temperature in the hair salons were satisfactory on the scale of the Ministry of Labor (Thailand). Conclusions:Installing and using proper ventilation in hair salon are recommended to eliminate health effects. Hairdresser worked in poor lighting that should be improved to appropriate levels (>1000 lux).

Synthesis of oligodeoxyribonucleotides containing a tricyclic thio analogue of O6-methylguanine and their recognition by MGMT and Atl1.

Promutagenic O6-alkylguanine adducts in DNA are repaired in humans by O6-methylguanine-DNA-methyltransferase (MGMT) in an irreversible reaction. Here we describe the synthesis of a phosphoramidite that allows the preparation of oligodeoxyribonucleotides (ODNs) containing a novel tricyclic thio analogue of O6-methylguanine in which the third ring bridges the 6-thio group and C7 of a 7-deazapurine. These ODNs are very poor substrates for MGMT and poorly recognised by the alkyltransferase-like protein, Atl1. Examination of the active sites of both MGMT and Atl1 suggest large steric clashes hindering binding of the analogue. Such analogues, if mutagenic, are likely to be highly toxic.

Measurements and health impacts of carbon black and BTEXs in photocopy centers.

The purpose of this study was to measure the concentration of carbon black and BTEXs at three photocopy centers and to assess the health risk following exposure. Air samples were collected by sampling in the breathing zone of workers during the class and examination periods. The results found that the concentration of carbon black before operation was higher than morning and afternoon in both class and examination periods. These levels are considerably lower than the suggested limited value set by OSHA. Toluene had the highest concentration but did not exceed of recommended exposure limit by NIOSH. The carbon black and toluene concentrations were higher in examination period than during the normal class period. The results from interview indicate that cough and sneeze are the highest symptom among workers. Carbon black and toluene concentrations in photocopy centers should be concerned in terms of indoor air quality and human health.

Synthesis of oligodeoxyribonucleotides containing a conformationally-locked anti analogue of O6-methyl-2'-deoxyguanosine and their recognition by MGMT and Atl1.

We show that DNA containing a conformationally-locked anti analogue of O(6)-alkylguanine is a poor substrate for human O(6)-methylguanine-DNA methyltransferase (MGMT) and the alkyltransferase-like protein, Atl1. This highlights the requirement for the syn conformation and rationalises why certain O(6)-alkylguanines are poor MGMT substrates.