Detection and characterization of DNA adducts of 3-methylindole.

The pneumotoxin 3-methylindole is metabolized to the reactive intermediate 3-methyleneindolenine which has been shown to form adducts with glutathione and proteins. Reported here is the synthesis, detection, and characterization of nucleoside adducts of 3-methylindole. Adducted nucleoside standards were synthesized by the reaction of indole-3-carbinol with each of the four nucleosides under slightly acidic conditions, which catalyze the dehydration of indole-3-carbinol to 3-methyleneindolenine. Following solid phase extraction, the individual adducts were infused via an electrospray source into an ion trap mass spectrometer for molecular weight determination and characterization of the fragmentation patterns. The molecular ions and fragmentation of the dGuo, dAdo, and dCyd adducts were consistent with nucleophilic addition of the exocyclic primary amine of the nucleosides to the methylene carbon of 3-methyleneindolenine. The apparent chemical preference of this addition lead primarily to dAdo and dGuo adducts, with substantially less of the dCyd adduct formed. No adduct with dThd was detected. The adducts were purified by HPLC and subsequent NMR analysis of the dGuo and dCyd adducts confirmed the proposed structures. Mass spectral fragmentation of the three adducts produced primarily two ions which were the result of the loss of either the 3-methylindole moiety or the sugar. On a triple quadrupole electrospray mass spectrometer, the neutral loss of the sugar, [M + H - 116](+), was utilized for selected reaction monitoring of the calf thymus DNA adducts, formed by incubations of 3-methylindole with various microsomes (rat liver, goat lung, and human liver). All three adducts were detected from each of the microsomal incubations, following extraction and cleavage of the DNA to the nucleoside level. The dGuo adduct was the primary adduct formed, with smaller amounts of the dAdo and dCyd adducts. Rat hepatocytes incubated with 3-methylindole produced the same three adducts, in approximately the same proportions, while no adducts were detected in untreated hepatocytes. Microsomal incubations in the presence of ([3-(2)H(3)]-methyl)indole confirmed the formation and identification of the adducts as well as the fragmentation patterns. These results demonstrate that bioactivated 3-methylindole forms specific adducts with exogenous or intact cellular DNA, and indicates that 3-methylindole may be a potential mutagenic and/or carcinogenic chemical.

Mechanism-based inactivation of cytochrome P450 2B1 by 7-ethynylcoumarin: verification of apo-P450 adduction by electrospray ion trap mass spectrometry.

7-Ethynylcoumarin was synthesized as a potential mechanism-based inhibitor, and it was found to be an effective inactivator of 7-ethoxy-4-(trifluoromethyl)coumarin (7EFC) O-deethylation catalyzed by purified, reconstituted P450 2B1. In contrast, 7-ethynylcoumarin demonstrated minimal inactivation of P450 2A6-mediated 7-hydroxycoumarin formation. The inactivation of P450 2B1 demonstrated pseudo-first-order kinetics and was NADPH- and inhibitor-dependent. The maximal rate constant for the inactivation of 2B1 was 0.39 min(-)(1) at 30 degrees C, and thus, the time required to inactivate 50% of the P450 2B1 that was present (t(1/2)) was 1.8 min. The estimated concentration which led to half-maximal inactivation (K(I)) was 25 microM. No protection from inactivation was seen in the presence of nucleophiles (glutathione and sodium cyanide), an iron chelator (deferroxamine), or superoxide dismutase and catalase. Addition of the substrate (7EFC) protected P450 2B1 from inactivation, in a concentration-dependent manner. The partition ratio for P450 2B1 was 25; i.e., the number of metabolic events was 25-fold higher than the number of inactivating events. Incubations of 7-ethynylcoumarin with P450 2B1 for 10 min resulted in an 80% loss in enzymatic activity, while 90% of the ability to form a reduced-CO complex remained. This activity loss was not recovered following dialysis, indicative of irreversible inactivation. Covalent attachment of the entire inhibitor and oxygen to apo-P450 2B1, in a 1:1 ratio, was shown via electrospray ion trap mass spectrometry. This method also verified the absence of modification to the heme or the cytochrome P450 reductase. Taken together, the characterization of the inhibition seen with P450 2B1 and 7-ethynylcoumarin was consistent with all of the criteria required to distinguish a mechanism-based inactivator. In addition, electrospray ion trap mass spectrometry has the potential to be applied to protein adducts above and beyond those associated with the mechanism-based inactivation of cytochrome P450s.

Orientation of caffeine within the active site of human cytochrome P450 1A2 based on NMR longitudinal (T1) relaxation measurements.

Longitudinal (T1) relaxation studies were performed in order to examine the interaction of caffeine with the heme of human P450 1A2. Addition of caffeine to this P450 resulted in a small, incomplete conversion of the heme from high spin to low spin, as shown by changes in the optical spectrum. Determination of a relatively large dissociation constant (Ks = 2.6 mM) as well as the relative instability of the P450 after 2 h at room temperature necessitated the performance of these experiments at high concentrations (25 mM) of caffeine. The relaxation measurements on the three sets of methyl hydrogens led to the determination of the corresponding distances between the iron and the methyl groups on the bound caffeine as well as the position and orientation of caffeine within the active site of P450 1A2. The three methyl groups were found to be nearly equidistant from the iron (> or = 4.79-4.89 A), with slight preference for the N-3 position, and thus, the average position of caffeine was parallel to the heme. In vitro incubations with P450 1A2 and 5 mM caffeine led primarily to paraxanthine formation (N-3 demethylation), as expected. However, with 25 mM substrate, the overall extent of oxidation was doubled and there was more equivalent oxidation at each of the four potential sites on caffeine. This latter observation was consistent with the lack of selective positioning of the N-3 methyl group of caffeine relative to the heme.

Subnanomolar quantification of caffeine's in vitro metabolites by stable isotope dilution gas chromatography-mass spectrometry.

A method for the quantification of subnanomolar levels of in vitro metabolites of caffeine by an isotope dilution gas chromatographic-mass spectrometric (GC-MS) assay has been developed and applied. Trideuteromethylated analogs of each primary metabolite were synthesized and added after incubations of caffeine with human liver microsomes high in cytochrome P4501A2. HPLC separation of the metabolites prior to GC-MS quantification allowed the isolation of theobromine and paraxanthine which coeluted by GC and enabled quantification over a larger dynamic range. Quantitative analysis was performed on the n-propylated derivatives by selected-ion monitoring of either the M+. ions for the dimethylxanthines or [M-C3H6]+. ions for 1,3,7-trimethyluric acid. For the least abundant metabolite (1,3,7-trimethyluric acid), the detection level on column was 200 pg. Replicate analyses exhibited intra- and inter-day variability of 4.2 and 7.9%, respectively. This assay has been successfully used in the quantification of caffeine's primary metabolites in more than 180 incubations, at varying substrate concentrations and with multiple enzyme sources.