Quantitative correlation of drug bioactivation and deoxyadenosine alkylation by acylfulvene.
Acylfulvenes (AFs) are a class of antitumor agents that exert their cytotoxic effects by forming covalent adducts with biomolecules, including DNA and proteins; clinical trials are ongoing for (-)-(hydroxymethyl)AF. Recently, depurinating DNA adducts N3-AF-deoxyadenosine (dAdo) and N7-AF-deoxyguanosine (dGuo) were identified from reactions of the parent compound, AF, with calf thymus DNA in the presence of the reductase enzyme alkenal/one oxidoreductase (AOR) and cofactor NADPH. We report here the development of a structure-specific quantitative analytical method for evaluating levels of the major base adduct N3-AF-adenine (Ade), which results from depurination of N3-AF-dAdo, and its utilization to further probe the relationship between AOR-mediated bioactivation and adduct formation in a cell-free system. As an internal standard, the isotopomer N3-AF-Ade-d3 was synthesized, and electrospray-ionization mass spectrometry coupled with high-performance liquid chromatography (HPLC-ESI-MS/MS) was used to detect and quantitate the adduct. This method was validated and found to be accurate (R2>or=0.99) and precise (relative standard deviation 5.8-6.4%), with a limit of detection of 2 fmol. DNA samples, to which the stable-isotope-labeled internal standard was added, were subjected to neutral thermal hydrolysis yielding N3-AF-Ade. Adducts were isolated by a simple solid-liquid methanol extraction procedure, and adduct formation was examined in the presence of either high (1-3 micromol) or low (15 nmol) levels of DNA. Absolute amounts of N3-AF-Ade were measured in cell-free reaction mixtures containing varying levels of AOR as the only drug-activating enzyme. The increase in adduct formation (5-100 adducts per 10(5) DNA bases) over a range of enzyme concentrations (1-24 nM of AOR) showed saturation type behavior. This study reports a sensitive HPLC-ESI-MS/MS method for quantitation of the major DNA adduct induced by AF and illustrates a correlation between N3-AF-Ade formation and AOR-mediated enzymatic activation in a cell-free system, thus providing a template for further studies of drug toxicity in cells and in vivo.
Neels
James F
JF
Department of Medicinal Chemistry and The Cancer Center, University of Minnesota, Minneapolis 55455, USA.
Gong
Jiachang
J
Yu
Xiang
X
Sturla
Shana J
SJ
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000478:Alkylation; D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D018736:DNA Adducts / Q000032:analysis / Q000378:metabolism*; D003839:Deoxyadenosines / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D015203:Reproducibility of Results; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013141:Spiro Compounds / Q000737:chemistry / Q000378:metabolism
D018906:Antineoplastic Agents, Alkylating; D018736:DNA Adducts; D003839:Deoxyadenosines; D012717:Sesquiterpenes; D013141:Spiro Compounds; C079422:acylfulvene; D000429:Alcohol Oxidoreductases