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Current
Drug Metabolism
ISSN: 1389-2002
Current Drug Metabolism
Volume 8, Number 1, January 2007
Contents
Chemoprotective and Carcinogenic Effects of tert
Butylhydroquinone and Its Metabolites Pp. 1-7
N. Gharavi, S. Haggarty and A.O.S. El-Kadi
[Abstract] [Full
Text Article]
Effects of Dietary Broccoli on Human in Vivo
Caffeine Metabolism: A Pilot Study on a Group of Jordanian
Volunteers Pp. 9-15
N. Hakooz and I. Hamdan
[Abstract] [Full
Text Article]
Hyperhomocysteinaemia: A Critical Review of Old and
New Aspects Pp. 17-31
W. Herrmann, M. Herrmann and R. Obeid
[Abstract] [Full
Text Article]
Scaling Factors for the Extrapolation of In Vivo
Metabolic Drug Clearance from In Vitro Data: Reaching
a Consensus on Values of Human Microsomal Protein and Hepatocellularity
Per Gram of Liver Pp. 33-45
Z.E. Barter, M.K. Bayliss, P.H. Beaune, A.R. Boobis, D.J.
Carlile, R.J. Edwards, J.B. Houston, B.G. Lake, J.C. Lipscomb,
O. Pelkonen, G.T. Tucker and A. Rostami-Hodjegan
[Abstract] [Full
Text Article]
The ABCA2 Transporter: Intracellular Roles in Trafficking
and Metabolism of LDL-Derived Cholesterol and Sterol-Related
Compounds Pp. 47-57
J.T. Mack, D.M. Townsend, V. Beljanski and K.D. Tew
[Abstract] [Full
Text Article]
Molecular Modeling-Guided Site-Directed Mutagenesis
of Cytochrome P450 2D6 Pp. 59-77
C. de Graaf, C. Oostenbrink, P.H.J. Keizers, B.M.A. van
Vugt Lussenburg, R.A.B. van Waterschoot, R.A. Tschirret-Guth,
J.N.M. Commandeur and N.P.E. Vermeulen
[Abstract] [Full
Text Article]
Influence of Phenobarbital on Morphine Metabolism
and Disposition: LC-MS/MS Determination of Morphine (M) and
Morphine-3-Glucuronide (M3G) in Wistar-Kyoto Rat Serum, Bile
and Urine Pp. 79-89
Y. Alnouti, M.K. Shelby, C. Chen and C.D. Klaassen
[Abstract] [Full
Text Article]
Abstracts
[Back to top]
Chemoprotective and Carcinogenic Effects of tert Butylhydroquinone
and Its Metabolites
N. Gharavi, S. Haggarty and A.O.S. El-Kadi
[Full
Text Article]
Tert-butylhydroquinone (tBHQ) has been commonly used as a
synthetic food antioxidant to prevent oils and fats from oxidative
deterioration and rancidity due to its potent anti-lipid peroxidation
activity. In North America, the maximum level of tBHQ allowed
in fat products is 0.02% with an acceptable daily intake of
0 – 0.7 mg/kg body weight. Extensive studies have demonstrated
that tBHQ exhibit anti-carcinogenic effect. The ability of
tBHQ to induce phase II xenobiotic metabolizing enzymes through
an Nrf2-dependent pathway is thought to be responsible for
the observed protective effect of tBHQ. It has been proposed
that tBHQ enhances Nrf2-mediated transcription by promoting
reactive oxygen species-mediated dissociation of Nrf2-Keap1,
Nrf2 stabilization, phosphatidylinositol 3-kinase (PI3K)/Akt
activity, and MAPK pathway activation. In contrast to the
beneficial effects of tBHQ, a number of studies have shown
that chronic exposure to tBHQ may induce carcinogenicity.
However, the precise mechanisms of tBHQ carcinogenicity are
not well understood. The toxicity or carcinogenicity of tBHQ
has been attributed to the formation of reactive GSH-conjugates,
generation of reactive species, CYP1A1 induction, caspase
activation and reduced GSH/ATP levels. This review provides
an account of recent mechanisms proposed for both chemoprotective
and carcinogenic effect of tBHQ.
[Back to top]
Effects of Dietary Broccoli on Human in Vivo
Caffeine Metabolism: A Pilot Study on a Group of Jordanian
Volunteers
N. Hakooz and I. Hamdan
[Full
Text Article]
Objectives: Induction or inhibition of cytochrome
P450 (CYP) enzyme activities, enzymes that activate or detoxify
xenobiotics, is one mechanism by which vegetables may alter
cancer risk. As the effect of food on CYP enzyme activities
have not been studied in the Jordanian population, we examined
the effect of supplementing the diet with broccoli on CYP1A2
and CYP2A6 activities.
Methods: Five men and five women, non-smokers, consumed
a standard diet of broccoli (500 g) for 6 days. Enzyme activities
were determined by measuring urinary metabolite ratios after
a 100 mg caffeine tablet on the seventh day.
Results: The mean CYP1A2 activity for men (21.1 ±3.2)
was significantly lower than that for women (27.6 ±1.6)
before the consumption of broccoli (P <0.05). These activities
were significantly induced in both men (52.5 ±6.6)
and women (36.6 ±8.4) after a standard diet of broccoli
(P <0.005). Similarly, the mean value of CYP2A6 activity
for men was 0.061 ±0.040 and for women, 0.144 ±0.039
before consumption of broccoli, which were significantly different
(P <0.05). The activity of CYP2A6 was induced in both groups
significantly after broccoli consumption (P <0.05). The
mean value for men was 0.193 ±0.02 and for women, 0.214
±0.064.
Conclusion: Our study on a group of Jordanians confirmed
the well-established observation that broccoli induces CYP1A2
activity. This study also demonstrates the effect of gender
and broccoli consumption on CYP2A6 activity in Jordanians.
[Back to top]
Hyperhomocysteinaemia: A Critical Review of Old and
New Aspects
W. Herrmann, M. Herrmann and R. Obeid
[Full
Text Article]
Hyperhomocysteinemia (HHCY) is a risk factor for cardiovascular
(CVD) and neurodegenerative diseases, osteoporotic fractures
and pregnancy complications. HHCY is common and is mostly
related to B-vitamin deficiency.
Retrospective and prospective studies emphasise the causal
relationship between HHCY and CVD risk. Some reported vitamin
intervention trials, however, did not demonstrate lower risk
of CVD after treatment. Confounding factors on the one hand
and low subject numbers on the other hand reduced the statistical
power of the results. Re-analysis of the VISP study (after
excluding renal failure and vitamin B12 status tampering factors),
detected a 21% decrease in the risk of stroke. This number
has been confirmed by results from the HOPE 2 vitamin intervention
trial. A significant decline of stroke-mortality (8 to 16
%) has been observed in the USA and Canada after fortification
of grain products with folate. Despite negative results from
secondary prevention trials regarding the CVD risk reduction
there is convincing evidence about the effectiveness of B-vitamin
supplementation in lowering the risk of stroke (approximately
20%). Additionally, HHCY was recently linked to the occurrence
and severity of chronic heart insufficiency. HHCY is also
a risk factor for osteoporotic fractures and vitamin treatment
can lower the fracture risk.
HHCY predicts the decline in cognitive function with age.
Hypomethylation is among the central mechanisms through which
HHCY may damage the brain. HHCY and low folate are causal
factors for pregnancy complications. In addition to the recommended
folate supplementation, vitamin B12 supplementation may also
decrease pregnancy complications.
[Back to top]
Scaling Factors for the Extrapolation of In Vivo
Metabolic Drug Clearance from In Vitro Data: Reaching
a Consensus on Values of Human Microsomal Protein and Hepatocellularity
Per Gram of Liver
Z.E. Barter, M.K. Bayliss, P.H. Beaune, A.R. Boobis, D.J.
Carlile, R.J. Edwards, J.B. Houston, B.G. Lake, J.C. Lipscomb,
O. Pelkonen, G.T. Tucker and A. Rostami-Hodjegan
[Full
Text Article]
Reported predictions of human in vivo hepatic clearance
from in vitro data have used a variety of values
for the scaling factors human microsomal protein (MPPGL) and
hepatocellularity (HPGL) per gram of liver, generally with
no consideration of the extent of their inter-individual variability.
We have collated and analysed data from a number of sources,
to provide weighted meangeo
values of human MPPGL and HPGL of 32 mg g-1 (95%
Confidence Interval (CI); 29 – 34 mg.g-1)
and 99 ×
106 cells.g-1 (95% CI; 74 – 131
mg.g-1), respectively.
Although inter-individual variability in values of MPPGL and
HPGL was statistically significant, gender, smoking or alcohol
consumption could not be detected as significant covariates
by multiple linear regression. However, there was a weak but
statistically significant inverse relationship between age
and both MPPGL and HPGL.
These findings indicate the importance of considering differences
between study populations when forecasting in vivo
pharmacokinetic behaviour. Typical clinical pharmacology studies,
particularly in early drug development, use young, fit, healthy
male subjects of around 30 years of age. In contrast, the
average age of patients for many diseases is about 60 years
of age. The relationship between age and MPPGL observed in
this study estimates values of 40 mg.g-1 for a
30 year old individual and 31 mg.g-1 for a 60 year
old individual. Investigators may wish to consider the reported
covariates in the selection of scaling factors appropriate
for the population in which estimates of clearance are being
predicted.
Further studies are required to clarify the influence of age
(especially in paediatric subjects), donor source and ethnicity
on values of MPPGL and HPGL. In the meantime, we recommend
that the estimates (and their variances) from the current
meta-analysis be used when predicting in vivo kinetic
parameters from in vitro data.
[Back to top]
The ABCA2 Transporter: Intracellular Roles in Trafficking
and Metabolism of LDL-Derived Cholesterol and Sterol-Related
Compounds
J.T. Mack, D.M. Townsend, V. Beljanski and K.D. Tew
[Full
Text Article]
ATP-binding cassette (ABC) transporters comprise a family
of critical membrane bound proteins functioning in the translocation
of molecules across cellular membranes. Substrates for transport
include lipids, cholesterol and pharmacological agents. Mutations
in ABC transporter genes cause a variety of human pathologies
and elicit drug resistance phenotypes in cancer cells. ABCA2,
the second member the A subfamily to be identified, was highly
expressed in ovarian carcinoma cells resistant to the anti-cancer
agent, estramustine, and more recently, in human vestibular
schwannomas. Cells expressing elevated levels of ABCA2 show
resistance to variety of compounds, including estradiol, mitoxantrone
and a free radical initiator, 2,2'-azobis-(2-amidinopropane).
ABCA2 is expressed in a variety of tissues, with greatest
abundance in the central nervous system and macrophages. This
transporter, along with other proteins that have a high degree
of homology to ABCA2, including ABCA1 and ABCA7, are up-regulated
in human macrophages during cholesterol import. Recent studies
have shown ABCA2 also plays a role in the trafficking of low-density
lipoprotein (LDL)-derived free cholesterol and to be coordinately
expressed with sterol-responsive genes. A single nucleotide
polymorphism in exon 14 of the ABCA2 gene was shown to be
linked to early onset Alzheimer disease (AD) in humans, supporting
an earlier study showing ABCA2 expression influences levels
of APP and β-amyloid
peptide, the primary component of senile plaques. Studies
thus far implicate ABCA2 as a sterol transporter, the deregulation
of which may affect a cellular phenotype conducive to the
pathogenesis of a variety of human diseases including AD,
atherosclerosis and cancer.
[Back to top]
Molecular Modeling-Guided Site-Directed Mutagenesis
of Cytochrome P450 2D6
C. de Graaf, C. Oostenbrink, P.H.J. Keizers, B.M.A. van
Vugt Lussenburg, R.A.B. van Waterschoot, R.A. Tschirret-Guth,
J.N.M. Commandeur and N.P.E. Vermeulen
[Full
Text Article]
Cytochrome P450 (CYP) 2D6 is one of the most important drug
metabolizing enzymes and the rationalization and prediction
of potential CYP2D6 substrates is therefore advantageous in
the discovery and development of new drugs. Experimentally,
the active site of CYP2D6 can be probed by site directed mutagenesis
studies. Such studies can be designed from structural models
of enzyme-substrate complexes. Modeling approaches can subsequently
be used to rationalize the observed effect of mutations on
metabolism and inhibition. The current paper will present
the construction, refinement and validation of the CYP2D6
homology model used in our laboratory for the prediction and
rationalisation of CYP2D6 substrate metabolism and CYP2D6-ligand
interactions. The model could explain reported site-directed
mutagenesis data (for example, mutation of E216 and D301).
Furthermore, based on the model, new CYP2D6 mutants were constructed
and studied in our lab, and also for these mutants a rationalization
of experimentally observed characteristics could be achieved
(I106E, F120A, T309V, F483A). CYP2D6-substrate interaction
fingerprint analysis of docked substrates in our homology
model suggests that several other active site residues are
probably interacting with ligands as well, opening the way
for further mutagenesis studies. Our homology model was found
to agree with most of the details of the recently solved substrate-free
CYP2D6 crystal structure [Rowland et al. J. Biol. Chem.
2006, 281, 7614-7622]. Structural
differences between the homology model and crystal structure
were the same differences observed between substrate-free
and substrate-bound structures of other CYPs, suggesting that
these conformational changes are required upon substrate binding.
The CYP2D6 crystal structure further validates our homology
modeling approach and shows that computational chemistry is
a useful and valuable tool to provide models for substrate-bound
complexes of CYPs which give insight into CYP-ligand interactions.
This information is essential for successful pre-experimental
virtual screening, as well as accurate hypothesis generation
for in vitro studies in drug discovery and development.
[Back to top]
Influence of Phenobarbital on Morphine Metabolism
and Disposition: LC-MS/MS Determination of Morphine (M) and
Morphine-3-Glucuronide (M3G) in Wistar-Kyoto Rat Serum, Bile
and Urine
Y. Alnouti, M.K. Shelby, C. Chen and C.D. Klaassen
[Full
Text Article]
A simple LC-MS/MS method has been developed and validated
for the simultaneous determination of morphine (M) and morphine-3-glucuronide
(M3G) in rat serum, bile, and urine. Deuterated D3-M
and D3-M3G were used as internal standards (IS)
for M and M3G, respectively. Serum samples were processed
by acetonitrile precipitation. Bile samples were prepared
by solid-phase extraction (SPE) using Oasis MCX cartridges.
Urine samples were directly analyzed after dilution with mobile
phase. Chromatography was performed using a Luna C18 column
(5 μm,
150 x 2.1 mm I.D.). The mobile phase consisted of acetonitrile
(ACN) and 7.5 mM ammonium formate (pH 9.3) delivered from
separate pumps with a simple gradient. The method was validated
to quantify M in the range of 1-1000 ng/ml in bile and serum,
and 0.025-25 μg/ml
in urine. M3G was quantified in the range of 1-1000 ng/ml
in serum, 0.1-100 μg/ml
in bile, and 0.05- 25 μg/ml
in urine. The method was applied to study the pharmacokinetics
and disposition of M and M3G in Wistar-Kyoto (WKY) rats, and
the effect of phenobarbital (PB) on M and M3G disposition.
M is metabolized to M3G at a lower rate in male than female
rats leading to higher M levels and lower M3G levels in serum,
urine, and bile of male than female rats. PB administration
induces M glucuronidation to M3G in male, but not female WKY
rats, and abolishes the gender differences in M and M3G pharmacokinetics.
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