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Accurate and sensitive quantitation of N7-methyldeoxyguanosine-3'-monophosphate by 32P-postlabeling and
K Haque1, D P Cooper, J H van Delft
1Cancer Research Campaign Department of Carcinogenesis and Medical Oncology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, U.K.
Abstract:
As N7-methyldeoxyguanosine-3'-monophosphate (N7-MedGp) is the major, persistent DNA lesion generated by methylating agents, a combined HPLC/32P-postlabeling assay has been developed to quantitate this adduct in human DNA. N7-MedGp was purified from normal nucleotides by anion-exchange chromatography followed by reverse-phase HPLC procedures. The adduct was then 32P-postlabeled and resolved by two-dimensional TLC for detection and quantitation by storage-phosphor imaging. The effect of conditions used for DNA purification and digestion on the recovery of N7-MedGp has been investigated. Extended, raised temperature incubations normally employed during DNA purification were demonstrated to result in considerable loss of adduct through depurination after 22 h at 65 and 37 degrees C (82% and 20% loss, respectively), but depurination was reduced to 5% if the incubation was performed at either 4 or 22 degrees C. Similarly, close to optical recovery (83%) of N7-MedGp was achieved after DNA digestion by incubating at 4 degrees C, pH 7.4, for 18 h in the presence of micrococcal nuclease and calf spleen phosphodiesterase from Sigma and Boehringer Mannheim, respectively. Overall, the recovery of N7-MedGp was 40%, resulting in a detection limit of 1.3 fmol which is equivalent to 0.16 mumol of adduct/mol of 2'-deoxyguanosine-3'-monophosphate (dGp) when analyzing 10 micrograms of DNA. The N7-MedGp content of DNA that had been methylated in vitro using 0, 16, and 80 microM N-methyl-N-nitrosourea (NMU) was determined by 32P-postlabeling to be 12, 112, and 671 mumol of N7-MedGp/mol of dGp. Electrochemical detection of N7-methylguanine (N7-MeG) after HPLC purification measured approximately 2-fold higher levels, i.e., 25, 225, and 1080 mumol of N7-MeG/mol of Gua, at each NMU concentration, respectively. The levels of N7-MedGp in the white blood cell (WBC) DNA of patients receiving a single dose of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC) chemotherapy were determined by 32P-postlabeling. Maximum levels were found 4-6 h after treatment, and in two out of four individuals adduct levels were decreased by 21 h. Prior to treatment, N7-MedGp was detectable in WBC DNA in two out of the four individuals indicating that nontherapeutic exposure to methylating agents had occurred.
Insights
A new HPLC/32P-postlabeling assay quantifies N7-methyldeoxyguanosine-3'-monophosphate (N7-MedGp), a key DNA lesion from methylating agents. Optimized conditions minimize adduct loss during DNA purification and digestion, enabling sensitive detection in human DNA.
Area of Science:
- Molecular Toxicology
- DNA Damage and Repair
- Analytical Chemistry
Background:
- N7-methyldeoxyguanosine-3 ext-monophosphate (N7-MedGp) is the primary persistent DNA adduct formed by methylating agents.
- Accurate quantification of N7-MedGp is crucial for understanding the genotoxic effects of methylating agents and assessing human exposure.
- Existing methods may be limited in sensitivity or affected by DNA processing conditions.
Purpose of the Study:
- To develop and validate a sensitive combined High-Performance Liquid Chromatography (HPLC) and 32P-postlabeling assay for quantifying N7-MedGp in human DNA.
- To investigate and optimize DNA purification and digestion conditions to maximize N7-MedGp recovery and minimize adduct loss.
- To determine N7-MedGp levels in DNA treated with N-methyl-N-nitrosourea (NMU) in vitro and in white blood cell (WBC) DNA from patients undergoing chemotherapy.
Main Methods:
- Development of a sequential anion-exchange and reverse-phase HPLC method for N7-MedGp purification.
- Application of 32P-postlabeling and two-dimensional Thin-Layer Chromatography (TLC) for adduct detection and quantification using storage-phosphor imaging.
- Systematic evaluation of DNA purification (temperature, time) and enzymatic digestion (temperature, pH, enzymes) conditions on N7-MedGp recovery.
Main Results:
- Optimized conditions (e.g., 4°C incubation) significantly reduced N7-MedGp loss due to depurination during DNA purification (5% loss vs. 82% at 65°C).
- High recovery (83%) of N7-MedGp was achieved during DNA digestion using specific enzymes at 4°C and pH 7.4.
- The assay achieved a detection limit of 1.3 fmol (0.16 µmol adduct/mol dGp) and quantified N7-MedGp adducts in NMU-treated DNA and patient WBC DNA, with levels correlating to NMU concentration and showing temporal changes post-chemotherapy.
Conclusions:
- The developed HPLC/32P-postlabeling assay is a sensitive and robust method for quantifying N7-MedGp in human DNA.
- Careful control of DNA purification and digestion conditions is essential to prevent artifactual adduct loss and ensure accurate measurement.
- The assay can detect endogenous and exogenously induced N7-MedGp adducts, providing a valuable tool for biomonitoring and genotoxicity studies.