radioactivity. The second portion (0.5 mL) of the last eluate is evaporated to
dryness under nitrogen overnight. The residue is reconstituted in 0.5 mL of
MeOH–H 2 O (2:1) and is counted for radioactivity. The difference in
radioactivity between prior and after evaporations is considered as a loss of
the amount of the tritium label. By dividing the amount of the tritium loss by
the radioactivity in the original sample, an extent of the tritium loss can be
estimated.
14.2.3 Protocol forIn vitroCovalent Protein Binding
in Human or Rat Hepatocytes
Cryopreserved human hepatocytes from three male and two female donors or
freshly isolated male rat hepatocytes are analyzed for viabilities (75–85%) using
the trypan blue exclusion methods. Incubations are performed by suspending the
hepatocytes in Krebs-biocarbonate buffer followed by addition of a^3 H-labeled
compound in methanol. The specific radioactivity of the compounds is 100 Ci/
mol. The final concentration of test compound in the suspension is 10mMina
final volume of 1 mL (1 106 cells/mL), and the final concentration of methanol
does not exceed 0.2% (v/v). Incubations are allowed to proceed at 37C for 1 h,
and are quenched with acetonitrile (5 mL). The remaining procedures are the
same as described in Section 14.2.2 (the protocol forin vitrocovalent protein
binding in human or rat liver microsomes – a test-tube method). Covalent
protein binding values in pmol-equiv./mg protein are estimated based on the
residual radioactivity in the protein pellets.
14.2.4 Protocol forIn vitroCovalent Protein Binding in Human
or Rat Liver Microsomes—A Semiautomated Method
A grand pool of suspension is prepared by mixing human or rat liver
microsomes, a^3 H-tracer of a test compound or a reference^3 H-tracer (Evans
et al., 2004) in phosphate buffer (100 mM, pH 7.4) containing EDTA (1 mM)
and MgCl 2 (0.1 mM). The specific radioactivity of the tracers is 100 Ci/mol.
Aliquots (160mL) of the suspension are transferred to individual wells of a 96-
well plate (Note 2). Reactions are initiated by adding 40mLofNADPH
solution (5 mM). Incubations in duplicate are performed at 37C with a final
protein concentration of 1 mg/mL, a test compound concentration of 10mM,
a NADPH concentration of 1 mM in a total volume of 200mL. Reactions are
quenched at 0 and 60 min by adding 800mL of acetone into individual wells
and are vortexed for 30 s. Proteins are collected by aspirating samples from the
96-well plate onto a prewet filter mat by a Brandel cell harvester (Brandel,
Gaithersburg, MD), and are washed with MeOH–H 2 O(8:2)for4times.
Individual filter disks are punched from the filter mat, and are placed in plastic
scintillation vials. One milliliter of 7.5% SDS is added to each vial. Caped vials
are placed in a water bath shaker at 55C and 200 rpm overnight. After the
vials are cooled to room temperature, an aliquot (20mL) of each sample in
464 PROTOCOLS FOR ASSESSMENT