Abstract:
The cytochrome P450 (CYP) enzyme superfamily is involved in the metabolism and elimination of many drugs. CYP2B6 is important in the metabolism of drugs such as bupropion, cyclophosphamide, and efavirenz. The formation of hydroxybupropion (OHBU) is selective for
CYP2B6 in humans, and bupropion is an FDA approved probe substrate for measuring
CYP2B6 activity clinically. There is a substantial inter-individual variation in CYP2B6 activity
due in part to inherited differences, as well as possible differences in expression between
males and females. This enzyme is highly inducible by environmental factors such as
medications and diet via the action of liver-enriched transcription factors such as constitutive
androstane receptor (CAR). The homologous murine enzyme is Cyp2b10, and recent
unpublished data has demonstrated a statistically significant (p < 0.0001) increase in hepatic
Cyp2b10 mRNA expression 4 hours after high intensity interval training (HIIT) exercise in
C57BL/6 male mice.
The aim of this thesis was to determine whether the observed increase in Cyp2b10 mRNA
post HIIT exercise resulted in a similar increase in hepatic Cyp2b10 protein expression and
activity.
Bupropion was used as the probe substrate, and an HPLC assay was optimized and validated
to FDA standards for accuracy and precision (< 15%) to quantify the formation of OHBU in
these livers. Since there was no prior literature on Cyp2b10 protein expression or the formation
of OHBU in C57BL/6 mice, initial studies were undertaken to compare hepatic metabolism in
CD-1 as well as C57BL/6 (male and female) mice. The in vitro incubation conditions for the
optimal formation of OHBU were determined and compared with the expected literature for
CD-1 female mice. The results indicated that CD-1 female mice had the highest and C57BL/6
male mice had the lowest intrinsic clearance of bupropion to OHBU.
The protein expression of Cyp2b10 in both strains was also determined using Western blotting,
using a commercially available Cyp2b10 antibody. C57BL/6 females had the lowest Cyp2b10
protein expression, which was not consistent with the data for OHBU formation.
The formation of OHBU, as a probe for Cyp2b10 activity, was then determined in liver samples
from the HIIT exercised mice. The activity was assessed at 50 μM bupropion substrate
concentration. There was no significant difference in activity across the individual HIIT
exercised mice (Rest vs 0, 2, 4, and 16h post exercise groups). However, the activity was
significantly (p < 0.0001) lower in all of these HIIT exercised mice compared to freshly
prepared C57BL/6 mouse liver microsomes. Analysis of full Michaelis-Menten kinetics (0-150
μM bupropion substrate) in the pooled samples from each treatment group (N=8 per group)
demonstrated significantly lower rates of OHBU formation at all timepoints post-HIIT
compared to the Rest group (p < 0.0001).
In contrast, there appeared to be a significant (p < 0.001) increase in Cyp2b10 protein
expression in HIIT exercised mice immediately after exercise (0 h group) compared to Rest
mice.
Preliminary studies were undertaken to determine if there were any changes in the hepatic
protein expression of Car, an important transcription factor that regulates the expression of
Cyp2b10 in mice. Multiple immunoreactive Car-related bands were detected in the hepatic
cytosol and a single band in microsomes. Changes in individual cytosolic Car-related proteins
were observed in HIIT exercised mice. Some of these Car-protein bands showed a statistically
significant increase after exercise compared to the Rest group, and some appeared to be
significantly decreased after exercise.
The results of these studies suggest that the HIIT exercise related change in Cyp2b10 mRNA
expression does not appear to correspond to a simple increase in Cyp2b10 protein or OHBU formation. However, this may be due to technical issues, such as the lack of an antibody
specific to murine Cyp2b10 since the commercially available antibody was raised against rat
Cyp2b1/b2 protein. In addition, the formation of OHBU in mouse liver, unlike the human liver,
may be catalyzed by multiple Cyp2b isoforms, and the formation of additional metabolites
(presumed to be threo-/erythro-hydrobupropion) could complicate the use of the formation of
OHBU as a selective probe of this isoform in mice. Future studies could increase the sample
timepoints of HIIT exercised mice, add the other sex group (female) and consider the selection
of a specific probe substrate for measuring Cyp2b10 activity.