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Kaivosaari et al.
divided into two families, UGT1 (or UGT1A) and UGT2, UGT2B10 (Girard et al., 2005), we set out to solve this
based on sequence homology and gene structure (Mackenzie enduring question regarding nicotine metabolism.
et al., 2005). The expression of UGTs is tissue-specific. How-
ever, most UGTs are expressed in more than one tissue, and
nearly each human tissue that contains UGTs carries more
than a single UGT isoform (Tukey and Strassburg, 2000;
Mackenzie et al., 2003). Nonetheless, the liver is the major
site of glucuronidation in our body, and most UGTs, but not
all, are expressed in liver cells.
The substrate-specificity of the human UGTs is highly
complex. Most human UGTs can glucuronidate several dif-
Materials and Methods
Materials. (Ϫ)-Nicotine hydrogen tartrate salt, (Ϫ)-cotinine, ala-
methicin (from Trichoderma viride), and D-saccharic acid 1,4-lactone
were purchased from Sigma-Aldrich (St. Louis, MO). UDPGA (as
triammonium salt) was from Fluka Chemie (Buchs, Switzerland).
Nicotine-N--glucuronide hydrate, nicotine-N--glucuronide(-meth-
yl-d3), cotinine-N--D-glucuronide, and (R,S)-cotinine-N--D-gluc-
uronide(-methyl-d3) were purchased from Toronto Research Chemi-
ferent compounds with variable chemical structure, albeit at cals (Toronto, ON, Canada). Pooled human liver microsomes (HLM;
18 donors) and pooled human intestine microsomes (HIM) were from
BD Biosciences (Bedford, MA). According to the manufacturer, the
intestine microsomes were prepared from both the duodenum and
jejunum sections of five different donors and so that they could be
regarded as small intestine microsomes. As a positive control for the
UGTs in the two different human microsomes samples, they were
subject to activity assays in the presence of 100 M. The entacapone
glucuronidation activity in HIM was high (27% of substrate was
converted to entacapone glucuronide using 0.3 mg/ml microsomal
protein activated with alamethicin, and 1-h incubation time), and it
different kinetics (Uchaipichat et al., 2004). This leads to
partial overlap in the substrate-specificity of individual
UGTs. However, alongside individual human UGTs that are
well-documented to metabolize many different aglycones
with high activity, there are also a few UGTs that so far have
demonstrated either no activity or only very limited activity
when screened against a large variety of aglycone substrates.
These so-called “orphan”, or nearly orphan, UGTs include
UGT2B10, UGT2B11 (Turgeon et al., 2003), UGT1A5 (Finel
et al., 2005), and perhaps UGT2B28 (Le´vesque et al., 2001). was 29% of the glucuronidation activity measured in HLM under the
same incubation conditions.
The UGTs mostly catalyze the conjugation of a hydroxyl
group in the aglycone substrate with glucuronic acid from the
cosubstrate UDPGA. However, some UGTs can also conju-
gate other functional groups such as different amines (Green
and Tephly, 1998; Hawes, 1998; Zenser et al., 2002; Borlak et
al., 2006). Nicotine is one of several xenobiotics that are
subjected to direct N-glucuronidation when incubated with
human liver microsomes in the presence of UDPGA
(Ghosheh et al., 2001). Which enzyme catalyzes nicotine glu-
Recombinant human UGTs were produced in baculovirus-infected
insect cells as described previously (Kurkela et al., 2003, 2007). The
relative expression levels of UGT1A4, UGT1A9, UGT2B7, and
UGT2B10 in the test samples were determined simultaneously by
immunodetection using a tetra-His monoclonal antibody (QIAGEN,
Hilden, Germany) as detailed elsewhere (Kurkela et al., 2007).
UGT mRNA Expression in Human Tissues. Total RNA was
extracted using TRIzol reagent (Sigma-Aldrich) from 47 human liv-
ers and pooled. Liver donors were all of European-American ancestry
curonidation? Several laboratories have tried to identify the and included both male (n ϭ 36) and female (n ϭ 11) subjects. Use of
the tissue was approved by the institutional review board of Tufts
University School of Medicine (Boston, MA). Total RNA from human
kidney (male African-American donor), small intestine (five Europe-
an-American donors), colon (three European-American donors), tra-
chea (male European-American donor), lung (male African-American
donor), and whole brain (male African-American donor) were pur-
chased from Clontech (Mountain View, CA), whereas total RNA from
human stomach (male European-American donor) was from Ambion
(Austin, TX). To generate cDNA, 1 g of total RNA was treated with
DNase enzyme (Promega, Madison, WI) and then reverse-tran-
main nicotine glucuronidating human UGT, but this under-
taking turned out to be highly challenging (Ghosheh and
Hawes, 2002b; Nakajima et al., 2002; Kuehl and Murphy,
2003). Among the tested human UGTs, UGT1A4, an enzyme
“specializing” in N-glucuronidation, was the most efficient in
cotinine glucuronidation (Kuehl and Murphy, 2003; Naka-
jima and Yokoi, 2005). However, it was also concluded that
the contribution of UGT1A4 to nicotine glucuronidation may
be minor, and another not-yet-identified UGT may play a
significant role in nicotine metabolism (Ghosheh and Hawes, scribed (SuperScript II; Invitrogen, Carlsbad, CA) with random hex-
amer primer (0.1 g) according to the manufacturers’ protocol. Quan-
titative PCR reactions (25 l) included Sybr Green 2X master mix
(Applied Biosystems, Foster City, CA), 10 l of 1:10 diluted cDNA
(except 1:30 dilution for liver cDNA and 1:500 to 1:1500 dilution for
cDNA assayed with 18S rRNA primers), and 200 nM concentration of
each primer. Primer pair sequences were as follows: CCC CTC GAT
GCT CTT AGC TGA GTG T (18S-rRNA, forward), CGC CGG TCC
AAG AAT TTC ACC TCT (18S-rRNA, reverse), TGC GCC ACA AAG
GAG CCA AAC AT (UGT2B10, forward), ATG ATA AAT AGC ACG
GTT GCC ACA CAA (UGT2B10, reverse), GTT ACG CTG GGC TAC
ACT CAA GG (UGT1A4, forward), CTC CAC ACA ACA CCT ATG
AAG GG (UGT1A4, reverse), TTT CAC AAG TAC AGG AAA TCA
TGT CAA T (UGT2B7, forward), and CAG CAG CTC ACT ACA GGG
AAA AAT (UGT2B7, reverse). Real-time PCR analysis (model 7300;
Applied Biosystems) was performed with the following PCR method:
95°C for 10 min, 40 to 45 cycles of 95°C for 30 s, and 60°C for 60 s.
Amplification specificity was ensured initially by sequencing of rep-
resentative PCR products, and in each run by PCR product duplex
melting-temperature analysis. Negative controls included the exclu-
sion of cDNA template and reverse-transcription enzyme. mRNA
concentrations were calculated using standard curves of PCR thresh-
old cycle number versus concentration of template, which were de-
2002b; Kuehl and Murphy, 2003; Nakajima and Yokoi, 2005).
Armed with an improved analytical method and prompted by
several incidental observations about the little-studied
Fig. 1. Chemical structures of nicotine, cotinine, and their respective
N-glucuronides.