medium is then collected, and the concentration of 6b-hydroxytestosterone, a
metabolite generated specifically by CYP3A4 is determined (Hariparsad et al.,
2004), thereby providing a specific quantitative measurement of CYP3A4-
dependent metabolic activity. The direct addition of a CYP3A4-specific
substrate to the culture media of whole cell monolayers, followed by
measurement of metabolite generation, is generally the most cost effective
and time efficient. Possible limitations of this whole-cell method are the
potential trapping of metabolites in the monolayers, which prevents their
release into the medium, and collateral effects of drug transporters and Phase II
enzymes on the overall rates of metabolite formation (LeCluyse, 2001b).
Alternatively, CYP3A4-dependent metabolic activity can also be determined
by harvesting hepatocytes, preparing microsomes, and measuring CYP3A4
activity in the microsomes (Luo et al., 2002). This method will accurately
measure CYP3A4 induction, but is time-consuming and usually not suitable
for high throughput screening. For most compounds, the observed fold
inductions compared to the vehicle control using intact monolayers and those
observed using isolated microsomal fractions are normally quite consistent
(LeCluyse, 2001b).
CYP3A4 protein expression can be measured directly by immunodetection
(LeCluyse et al., 2000; Luo et al., 2002). Microsomal protein (3mg) is resolved
by SDS–polyacrylamide gel electrophoresis (12% acrylamide). Resolved
proteins are transferred to nitrocellulose membranes, which are incubated in
3% bovine serum albumin in phosphate-buffered saline supplemented with
Tween 20 (0.1 M, pH 7.4, 0.1% Tween 20) for 45 min to block nonspecific
protein binding. Membranes are then treated with primary antiCYP3A4
antibody (Gentest, Woburn, MA), followed by horseradish peroxidase-
conjugated antimouse secondary antibody. The antibody-reactive CYP3A4
protein bands are visualized using enhanced chemiluminescence detection and
quantitated by photodensitometry (Desai et al., 2002).
For Northern Blot Analysis of CYP3A4 mRNA, total cellular RNA is
isolated as described using TRIzol reagent (Invitrogen, Carlsbad, CA) (Desai
et al., 2002). The amount of RNA is estimated by the absorbance ratio at
260/280 nm. A 10-mg aliquot is fractionated by electrophoresis in 1% agarose
gels containing formaldehyde (2.2 M) and transferred onto a nylon membrane
(Millipore Corp., Bedford, MA). The amount of mRNA loaded per lane is
verified and normalized by ethidium bromide staining of 18S and 28S rRNA,
which is visualized and photographed under UV illumination. The membranes
are hybridized with a CYP3A4-specific cDNA probe (780-base pair; Oxford
Biomedical Research, Inc., Oxford, MI) labeled with [^32 P]dCTP (PerkinElmer
Life Sciences, Boston, MA) using the random primer method (Desai et al.,
2002). CYP3A4 mRNA is quantitated by scanning densitometry of the
autoradiographs and normalized againstb-actin mRNA (Luo et al., 2002).
Alternative assays for quantitation of CYP3A4 mRNA (RT–PCR and bDNA)
are described in detail in recent publications (Czerwinski et al., 2002;
Prueksaritanont et al., 2005).
ASSESSMENTS 555