Materials and Methods

Reagents

Activated charcoal (untreated powder, 100–400 mesh; cat. no. C-5260; lot no. 54H0279) was obtained from the Sigma Chemical Co. (St. Louis, MO). The charcoal was freshly prepared as a suspension (0.8% w/v) in 0.1 M potassium phosphate buffer (pH 7.4) and was stirred continuously while in use. [1-¹⁴C]Ethyl iodide (50 mCi/mmol) and [1, 2-¹⁴C]acetaldehyde (110 mCi/mmol) were purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). All other reagents were purchased commercially at the best obtainable grade. Human B-lymphoblastoid microsomes containing cDNA-expressed CYP protein were purchased from Gentest Corp. (Woburn, MA). Human liver microsomes (N = 11 subjects) were prepared from organ donor tissue and were characterized as previously described (3, 4, 16, 17).

Synthesis of [O-Ethyl ¹⁴C]Phenacetin

[1-¹⁴C]Ethyl iodide (5.3 mCi, 0.1 mmol) was transferred to methyl ethyl ketone (0.2 mL) under vacuum. Acetaminophen (23 mg, 0.15 mmol) and powdered anhydrous potassium carbonate (28 mg, 0.2 mmol) was added in a mini reflux apparatus. [1-¹⁴C]Ethyl iodide in methyl ethyl ketone (0.2 mL) was then added to the mini reflux apparatus and rinsed with more methyl ethyl ketone (0.3 mL). The suspension was heated to reflux for 8 hours. The reaction mixture was then diluted with water (2 mL) and extracted three times with methylene chloride (1 mL). The organic layers were combined and washed with 1 N sodium hydroxide (2 mL), brine (2 mL), and water (2 mL) to remove any unreacted starting materials. The clear methylene chloride solution was dried over magnesium sulfate, filtered, and concentrated to dryness in vacuo. The residue (4.17 mCi, 79%) was dissolved in ethanol (2.5 mL), chilled, and filtered once again, and the radiochemical purity of the final product (> 99.5%) was determined by radio-thin layer chromatography on silica gel (Silica Gel 60, 5 × 20 cm, 250 μm thick; EM Separations Technology, Gibbstown, NJ), by using a Radiomatic Model RA radio-thin layer chromatography scanner (Packard Instrument Co., Meriden, CT). Methylene chloride: ethyl acetate (20:80, v/v) was employed as the solvent system, and the final radiolabeled product and authentic phenacetin standard were similarly eluted (R_F = 0.54). Mass spectrometry data confirmed this material to be [O-ethyl ¹⁴C]phenacetin, since the spectra and pseudo molecular ion (m/z at 180 amu for [M + H]⁺) were identical to that of authentic phenacetin standard. The m/z 180 amu: m/z 182 amu abundance ratio indicated that approximately 88% of the sample was labeled with a single carbon-14 atom. This corresponded to a specific activity of about 55 mCi/mmol, in reasonable agreement with the manufacturers specifications for the [1-¹⁴C]ethyl iodide. The final working solution of [O-ethyl ¹⁴C]phenacetin was 33.4 mM (1.67 mCi/mL ethanol; 50 mCi/mmol) and was used for all subsequent experiments. In some cases, unlabeled phenacetin was added to the solution to obtain a lower specific activity.

PODase Activity

Incubations were carried out in 2.0-mL polypropylene microcentrifuge tubes (ClickSeal^TM microtubes, Research Products International Corp., Mount Prospect, IL) in a Dubnoff shaking water bath at 37 °C (under air). Briefly, the final assay volume was 0.5 mL and consisted of the following at the indicated final concentration: 0.1 M potassium phosphate buffer (pH 7.4), EDTA (0.1 mM), MgCl₂ (3.0 mM), microsomal protein (0.25–1.0 mg/mL), and [O-ethyl¹⁴C]phenacetin (1–100 μM, 50 mCi/mmol; 200 μM, 17.2 mCi/mmol; added in 2 μL of ethanol, 0.4%, v/v). After a 3-min preincubation period, the reaction was initiated by addition of a NADPH-generating system containing NADP⁺ (4.0 mM), D-glucose 6-phosphate (10 mM) and D-glucose 6-phosphate dehydrogenase (Sigma Type VII, from baker’s yeast, 2.0 U/mL). Incubations with human B-lymphoblastoid microsomes containing cDNA-expressed CYP1A2 (133 pmol CYP1A2/mg protein) were carried out as described for native human liver microsomes, except that the reactions were started by addition of rapidly thawed microsomal protein (final assay concentration was 0.25 mg/mL; 33 pmol CYP1A2/mL) (3). No metabolism was detected in microsomes prepared from B-lymphoblastoid cells devoid of CYP.

At the required time, the reaction was terminated with 50 μL of 1 M HCl, and the samples were placed on ice for at least 5 min. After thorough vortexing, the charcoal suspension (0.45 mL) was added and the sample was vortexed once again. All samples were immediately centrifuged at 4°C (16,000 g, 10 min). Thereafter, 0.5 mL of supernatant was mixed with 15 mL of Ultima Gold-XR scintillation cocktail (Packard Instrument Co., Meriden, CT), and the samples were analyzed for [¹⁴C]acetaldehyde by radioassay. The majority of the added [¹⁴C]phenacetin (> 99%) was bound to charcoal.

Although the further oxidation of acetaldehyde, to acetic acid and/or CO₂, was not addressed, the recovery of total radioactivity in the supernatant was 90 ± 4% (mean ± SD of over 30 experiments performed during a 4-month period) after incubation of [1, 2-¹⁴C]acetaldehyde (0.05 μM to 50 μM) with human liver microsomes and a NADPH-generating system (≤ 40 min, 37 °C). In addition, no metabolism of [O-ethyl¹⁴C]phenacetin was detected in the absence of a NADPH-generating system.

The quantity of acetaldehyde formed was calculated from the net dpm observed (dpm in supernatant at time t minus dpm in supernatant at time zero), corrected to the total volume of the supernatant (0.5 mL aliquot/1.0 mL supernatant), and corrected for daily acetaldehyde recovery. The total net dpm’s were then converted to nanomoles of product from the specific activity of the substrate (typically 111,000 dpm/nmol) (3, 16). Estimates of apparentK_m and V_maxwere obtained by using PCNONLIN (3). The kinetic parameters were determined under linear reaction conditions (≤ 40 min; 0.25–1.0 mg protein/mL) over a substrate concentration range of 1–200 μM.

Inhibition Studies.

The inhibition of PODase activity was investigated using a number of purported CYP form selective inhibitors. Except for 4-methylpyrazole and diethyldithiocarbamate (dissolved in water), all inhibitors were dissolved in ethanol. The final volume of ethanol in the assay did not exceed 1.0% (v/v). In all cases, the concentrations chosen exceeded (≥ 10-fold) apparent K_i and/orK_m (3, 4, 9, 11, 14).