Roles of rat and human aldo-keto reductases in metabolism of farnesol and geranylgeraniol

Article abstract of DOI:10.1016/j.cbi.2010.12.017

Farnesol (FOH) and geranylgeraniol (GGOH) with multiple biological actions are produced from the mevalonate pathway, and catabolized into farnesoic acid and geranylgeranoic acid, respectively, via the aldehyde intermediates (farnesal and geranylgeranial). We investigated the intracellular distribution, sequences and properties of the oxidoreductases responsible for the metabolic steps in rat tissues. The oxidation of FOH and GGOH into their aldehyde intermediates were mainly mediated by alcohol dehydrogenases 1 (in the liver and colon) and 7 (in the stomach and lung), and the subsequent step into the carboxylic acids was catalyzed by a microsomal aldehyde dehydrogenase. In addition, high reductase activity catalyzing the aldehyde intermediates into FOH (or GGOH) was detected in the cytosols of the extra-hepatic tissues, where the major reductase was identified as aldo-keto reductase (AKR) 1C15. Human reductases with similar specificity were identified as AKR1B10 and AKR1C3, which most efficiently reduced farnesal and geranylgeranial among seven enzymes in the AKR1A-1C subfamilies. The overall metabolism from FOH to farnesoic acid in cultured cells was significantly decreased by overexpression of AKR1C15, and increased by addition of AKR1C3 inhibitors, tolfenamic acid and R-flurbiprofen. Thus, AKRs (1C15 in rats, and 1B10 and 1C3 in humans) may play an important role in controlling the bioavailability of FOH and GGOH.

Full text of DOI:10.1016/j.cbi.2010.12.017

Chemico-Biological Interactions 191 (2011) 261–268
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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
Roles of rat and human aldo–keto reductases in metabolism of farnesol and
geranylgeraniol
Satoshi Endoa,b,∗, Toshiyuki Matsunagaa, Chisato Ohtaa, Midori Sodaa, Ayano Kanamoria,
Yukio Kitadeb, Satoshi Ohnoc, Kazuo Tajimad, Ossama El-Kabbanie, Akira Haraa
a Gifu Pharmaceutical University, Gifu 501-1196, Japan
b United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu 501-1193, Japan
c Faculty of Engineering, Gifu University, Gifu 501-1193, Japan
d Hokuriku University, Kanazawa 920-1181, Japan
e Monash Institute of Pharmaceutical Sciences, Victoria 3052, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 25 December 2010
Farnesol (FOH) and geranylgeraniol (GGOH) with multiple biological actions are produced from the
mevalonate pathway, and catabolized into farnesoic acid and geranylgeranoic acid, respectively, via the
aldehyde intermediates (farnesal and geranylgeranial). We investigated the intracellular distribution,
sequences and properties of the oxidoreductases responsible for the metabolic steps in rat tissues. The
oxidation of FOH and GGOH into their aldehyde intermediates were mainly mediated by alcohol dehy-
drogenases 1 (in the liver and colon) and 7 (in the stomach and lung), and the subsequent step into
the carboxylic acids was catalyzed by a microsomal aldehyde dehydrogenase. In addition, high reduc-
tase activity catalyzing the aldehyde intermediates into FOH (or GGOH) was detected in the cytosols of
the extra-hepatic tissues, where the major reductase was identified as aldo–keto reductase (AKR) 1C15.
Human reductases with similar specificity were identified as AKR1B10 and AKR1C3, which most effi-
ciently reduced farnesal and geranylgeranial among seven enzymes in the AKR1A-1C subfamilies. The
overall metabolism from FOH to farnesoic acid in cultured cells was significantly decreased by overex-
pression of AKR1C15, and increased by addition of AKR1C3 inhibitors, tolfenamic acid and R-flurbiprofen.
Thus, AKRs (1C15 in rats, and 1B10 and 1C3 in humans) may play an important role in controlling the
bioavailability of FOH and GGOH.
Keywords:
Alcohol dehydrogenase
Aldehyde dehydrogenase
AKR1C15
AKR1C3
Farnesol
Geranylgeraniol
© 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Farnesol (FOH) and geranylgeraniol (GGOH) are produced by
dephosphorylation of farnesyl pyrophosphate and geranylgeranyl
Isoprenoids are intermediates of the mevalonate pathway that
produces cholesterol, ubiquinone, dolichol, heme a, and the 15-
carbon farnesyl and 20-carbon geranylgeranyl groups of prenylated
proteins [1–3]. Prenylation, a post-translational addition of the
isoprenyl moieties to proteins from farnesyl pyrophosphate and
geranylgeranyl pyrophosphate, has been shown to be crucial for
the biological functions of prenylated proteins in the regulation of
cell proliferation, differentiation, apoptosis and cytoskeleton orga-
nization.
pyrophosphate, respectively, although they are in turn phospho-
rylated to their pyrophosphates [3–5]. The two isoprenoids are
increasingly known to display multiple functions. FOH was shown
to exhibit diverse biological actions such as inhibition of Ca2+
-
channels [6] and activation of farnesoid X receptor [3], constitutive
androstane receptor [7] and peroxisome proliferator-activated
receptors ␣ and ␥ [8]. GGOH was shown to be a ligand for perox-
isome proliferator-activated receptors ␣ and ␥ [8] and a negative
regulator of liver X receptor ␣ [9], and its constant synthesis in
mouse brain was suggested to be essential for learning [10].
FOH is oxidized into farnesal (FAL), which is then metabo-
lized into farnesoic acid (FA) and prenyl dicarboxylic acids [11,12].
Since no biological action has been found for the metabolites, this
metabolism is thought to be a catabolic pathway (Fig. 1). Horse
and human alcohol dehydrogenases (ADHs) catalyze the oxidation
of FOH [13,14], but the enzyme responsible for the oxidation of
FAL into FA remains unknown. GGOH is also converted into ger-
anylgeranoic acid (GGA) and 2,3-dihydrogeranylgeranoic acid via
geranylgeranial (GGAL) in rat cultured cells [15]. Distinct from the
Abbreviations: ADH, alcohol dehydrogenase; AKR, aldo–keto reductase; ALDH,
aldehyde dehydrogenase; FA, farnesoic acid; FAL, farnesal; FAL-DH, FAL dehy-
drogenase; FAL-R, FAL reductase; FOH, farnesol; FOH-DH, FOH dehydrogenase;
GGA, geranylgeranoic acid; GGAL, geranylgeranial; GGAL-DH, GGAL dehydrogenase;
GGAL-R, GGAL reductase; GGOH, geranylgeraniol; GGOH-DH, GGOH dehydroge-
nase; MW, molecular weight; RT, reverse transcription; U, unit.
∗
Corresponding author at: Laboratory of Biochemistry, Gifu Pharmaceutical Uni-
versity, 5-6-1 Daigaku-Nishi, Gifu 501-1196, Japan. Tel.: +81 58 230 8100.
E-mail address: sendo@gifu-pu.ac.jp (S. Endo).
0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2010.12.017

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2.2. Assay of enzyme activity
Dehydrogenase activity was assayed by measuring the rate of
change in NADH absorbance (at 340 nm) or its fluorescence (at
455 nm with an excitation wavelength of 340 nm) in a 2.0-ml
reaction mixture containing 0.1 M potassium phosphate, pH 7.4,
2 mM NAD+, substrate and enzyme. The activities of ADH, FOH-DH,
GGOH-DH, FAL-DH and GGAL-DH were determined with 40 mM
ethanol, 0.1 mM FOH, 50 ␮M GGOH, 50 ␮M FAL and 20 ␮M GGAL,
respectively, as the substrates. In the assay of FAL-R and GGAL-R
activities, 0.1 mM NADPH and 50 ␮M FAL or 20 ␮M GGAL were used
as the coenzyme and substrate, respectively, in the above reaction
mixture, and the rate of NADPH oxidation was spectrophotomet-
rically determined. The dehydrogenase and reductase rates were
corrected for the non-enzymatic NAD+ reduction and NADPH oxi-
dation, respectively, by the enzyme samples in the reaction mixture
without the substrate. One unit (U) of enzyme activity was defined
as the enzyme amount that catalyzes the formation or oxidation of
1 ␮mol of NAD(P)H per minute at 25 ◦C. The apparent Km and Vmax
values were determined using five concentrations of the substrates
by fitting the initial velocities to the Michaelis–Menten equation,
and the kcat values were calculated from the Vmax values assuming
the molecular masses of ADH subunit and AKRs as 40 and 36 kDa,
respectively. Kinetic constants and IC50 values are expressed as the
means of two or three determinations.
Fig. 1. Metabolic pathways of FOH and GGOH. Lyase is lysosomal prenylcysteine
lyase that releases FAL and GGAL from prenylated proteins, and the other enzymes
shown in italic letters are studied in the present work.
FOH metabolites, the two carboxylic acid metabolites of GGOH
exhibit biological actions, such as induction of apoptosis in hep-
atoma cells [16], differentiation of mouse osteoblasts [17], and
formation of lipid droplets in HL-60 cells [18]. However, there has
been no report on the enzymes responsible for the metabolism of
GGOH into its metabolites.
Recently, a lysosomal prenylcysteine lyase that releases FAL and
GGAL from prenylated proteins was identified, indicating another
metabolic pathway that provides the two aldehyde metabolites
[19,20]. In addition, FAL was reported to be efficiently reduced
by AKR1C15, a rat NADPH-dependent reductase belonging to the
aldo–keto reductase (AKR) superfamily [21], proposing the pres-
ence of a novel metabolic pathway by which FAL is salvaged
into the biologically active FOH. However, it remains unknown
whether AKR1C15 reduces GGAL, and how the salvage pathway
contributes to the metabolism of FOH and GGOH. In order to iden-
tify the enzymes in the catabolism and proposed salvage pathway
of FOH and GGOH, we investigated their intracellular distribution,
amounts and properties in rat tissues and cultured cells. In this
paper, we designate the enzymes in the metabolic steps (Fig. 1) as
FOH dehydrogenase (FOH-DH), GGOH dehydrogenase (GGOH-DH),
FAL dehydrogenase (FAL-DH), GGAL dehydrogenase (GGAL-DH),
FAL reductase (FAL-R) and GGAL reductase (GGAL-R). The results
show the importance of the salvage pathway mediated by FAL-R
and GGAL-R for controlling the concentrations of FOH and GGOH,
and reveal that AKR1C15 is the major enzyme exhibiting both FAL-
R and GGAL-R activities in rat extra-hepatic tissues. Therefore, we
also examined human enzymes that mediate the salvage path-
way.
2.3. Product identification
The reaction was conducted in a 2.0-ml system containing 1 mM
NADPH or 2 mM NAD+, substrate (50 ␮M FAL, 20 ␮M GGAL, 50 ␮M
GGOH or 0.1 mM FOH), enzyme and 0.1 M potassium phosphate, pH
7.4. The substrate and products were extracted into 4 volumes of
ethyl acetate after 30 min-incubation at 37 ◦C, and were analyzed
by TLC as described previously [24].
2.4. Preparation of tissue samples and gel filtration
Tissues were excised from 16 to 20 week-old female Wistar rats.
The livers, lungs and stomachs (each 4 g) were homogenized with 9
volumes of 20 mM Tris–HCl, pH 7.5, containing 0.25 M sucrose, and
the homogenates were centrifuged at 700 × g for 10 min. The mito-
chondrial, microsomal and cytosolic fractions were prepared from
the supernatants by differential centrifugation, and the proteins
of the mitochondrial and microsomal fractions were solubilized as
described previously [25]. In the gel-filtration analysis, the extracts
of the rat tissues (10 g) were prepared by the centrifugation of
the homogenates at 9000 × g for 10 min, and subjected to ammo-
nium sulfate fractionation (30–80% saturation). The precipitated
proteins were dissolved into 10 ml of buffer A (10 mM Tris–HCl,
pH 8.0, plus 2 mM 2-mercaptoethanol), and applied to a Sephadex
G-100 column (3 × 90 cm) that has been equilibrated with the
same buffer. The FAL-R and GGAL-R activities in the fractions
were determined with 0.1 M potassium phosphate, pH 6.0, contain-
ing 1.0 mM 4-methylpyrazole as the assay buffer to eliminate the
reductase activity due to ADH. The fractions with FOH/GGOH-DH
and FAL/GGAL-R activities were separately pooled and concen-
trated by ultrafiltration. All procedures including homogenization
and gel filtration were carried out at 4 ◦C. Protein concentration
was determined by a bicinchoninic acid protein assay reagent kit
(Pierce) using bovine serum albumin as the standard.
2. Materials and methods
2.1. Chemicals
All-trans-forms of FOH, FAL, GGOH and GGA were purchased
from Sigma–Aldrich, Fluka, Wako Chemicals (Osaka, Japan) and
Biomol, respectively. trans,trans-GGAL was synthesized from GGOH
as described previously [22]. trans,trans-FA was prepared by
incubation of 0.1 mM FAL with 2 units (U) baker’s yeast alde-
hyde dehydrogenase (ALDH, Sigma–Aldrich) in 0.1 M potassium
phosphate buffer, pH 8.0, containing 2 mM NAD+ at 25 ◦C for
12 h. [1-14C] trans,trans-GGOH and [1-14C] trans,trans-FOH were
obtained from American Radiolabeled Chemicals (St. Louis, MO),
and resins for column chromatography were from GE Healthcare.
An AKR1C1 inhibitor, 3-bromo-5-phenylsalicylic acid, was synthe-
sized as described [23]. All other chemicals were of the highest
grade that could be obtained commercially.
2.5. cDNA isolation
The cDNAs for ADH1 and ADH7 were isolated from the
total RNA preparations of rat liver and stomach, respectively,
by reverse transcription (RT)-PCR. The DNA techniques fol-

S. Endo et al. / Chemico-Biological Interactions 191 (2011) 261–268
263
lowed the standard procedures described by Sambrook et al.
[26]. PCR was performed with Pfu DNA polymerase (Stratagene)
and the following pairs of sense and antisense primers: 5ꢀ-
atgagcacagctggaaaagta-3ꢀ and 5ꢀ-catgaatgccttcccggttt-3ꢀ for ADH1
cDNA; and 5ꢀ-atggacactgctggaaaag-3ꢀ and 5ꢀ-cagctctctggatctcaaa-3ꢀ
for ADH7 cDNA. The PCR products were ligated into pCR T7/CT-
TOPO vectors (Invitrogen), and the expression constructs were
transfected into Escherichia coli BL21 (DE3) pLysS (Invitrogen). The
inserts of the cloned cDNAs were sequenced by using a Beckman
CEQ2000XL DNA sequencer.
(Takara, Kusazu, Japan) and the gene-specific primers for ADH1 and
ADH7 as described above. The cDNA for ALDH3A2 was amplified
using sense and antisense primers, 5ꢀ-atggagcgacaggtccaacg-3ꢀ and
5ꢀ-gtttcctgtgtagagaatgtg-3ꢀ. The RT-PCR analyses for the expression
of mRNAs for AKR1C15 [21] and AKR1C9 [27] were carried out as
described previously.
2.9. Immunochemical experiments
Antibodies against the purified recombinant ADH7 and AKR1C3
were raised in rabbits, and their immunoglobulin fractions were
prepared from the antiserum. Immunoprecipitation of ADH7 and
AKR1C15 from preparations of purified enzymes and cell extracts
using the antibodies against ADH7 and AKR1C15 were performed as
described previously [21]. In Western blotting analyses using these
antibodies, the immunoreactive proteins were visualized using an
enhanced chemi-luminescence substrate system (GE Healthcare).
2.6. Enzyme purification
The recombinant ADH1 and ADH7 were expressed in the E. coli
cells, which were cultured in a LB medium containing ampicillin
(50 ␮g/ml) for 24 h at 20 ◦C after the addition of 1 mM isopropyl-
␤-d-thiogalactopyranoside. The enzymes were purified at 4 ◦C
from the extracts of the E. coli cells, which were obtained by
centrifugation (at 12,000 × g for 15 min) after sonication. In the
purification of ADH1, the cell extract was dialyzed against buffer
B (10 mM Tris–HCl, pH 8.5, plus 5 mM 2-mercaptoethanol), and
applied to a Q-Sepharose column (2 × 20 cm). The enzyme, eluted
in the non-adsorbed fraction, was applied to a Blue-Sepharose col-
umn (2 × 10 cm). The enzyme was eluted with a linear gradient
of 0–0.1 M NaCl in buffer A containing 1 mM NAD+. The enzyme
fractions were concentrated by ultrafiltration, and gel-filtrated on
a Sephadex G-200 column (3 × 90 cm) that had been equilibrated
with buffer A. The obtained enzyme showed a single 40 kDa pro-
tein band on SDS-PAGE analysis. In the purification of ADH7, the
cell extract was applied to a DEAE-Sephacel column (2 × 20 cm)
after dialysis against buffer C (10 mM Tris–HCl, pH 8.0, plus 5 mM
2-mercaptoethanol). The enzyme was eluted from the column with
a linear gradient of 0–0.15 M NaCl in buffer C. The enzyme frac-
tion was applied to the Blue-Sepharose column, and the adsorbed
enzyme was eluted with buffer C containing 1 mM NAD+. The
homogeneous preparation of ADH7 was obtained by gel-filtration
on the Sephadex G-200 column. Recombinant rat AKRs (1C9 and
1C15) [21,27] and human AKRs (1A1, 1B1 [28], 1B10 [24], 1C1, 1C4
[29], 1C2 [30] and 1C3 [31]) were expressed from their cDNAs, and
purified to homogeneity (judged by SDS-PAGE) as described.
The FOH-DHs of rat liver and stomach were purified from the
high-molecular weight (MW) enzyme fractions in the gel filtration
analyses of the two tissue extracts, according to the procedures
for purification of recombinant ADH1 and ADH7, respectively. The
hepatic FAL-R was purified from the low-MW enzyme fraction in
the gel filtration analysis of the tissue extract. The fraction was dia-
lyzed against buffer D (10 mM Tris–HCl, pH 8.0, containing 5 mM
2-mercaptoethanol and 20% glycerol), and applied to a Q-Sepharose
column (2 × 10 cm). The enzyme was eluted with a linear gradient
of 0–0.1 M NaCl in buffer D, and applied to a Red-Sepharose column
(1.2 × 5 cm). The adsorbed enzyme was eluted from the column
with buffer D containing 0.5 mM NADP+.
2.10. Cell culture experiments
Rat gastric mucosa RGM1 cells (RIKEN Cell Bank, Tsukuba, Japan)
were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F12
(1:1) combined medium supplemented with 10% (v/v) fetal bovine
serum, penicillin (100 U/ml), and streptomycin (0.1 mg/ml) at 37 ◦C
in a humidified incubator containing 5% CO2. HeLa and MCF7 cells
(ATCC, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s
medium plus 10% fetal bovine serum and the antibiotics. The trans-
fection of the anti-ADH7 antibodies (10 ␮g/ml of the medium)
into the cells was performed with a protein transfection reagent,
Profect P-2 (Targeting systems, Santee, CA), according to the man-
ufacturer’s manual. The transfected cells were maintained in the
medium for 24 h, and then used for experiments.
The pGW1 expression vectors harboring the cDNAs for AKR1B10
and AKR1C15 were constructed as described [24,33]. Similarly, the
expression vector harboring the cDNA for AKR1C3 was prepared.
The cDNA was initially amplified from the bacterial expression
vectors [31] by PCR using the primers. The sense primer, 5ꢀ-
ttgaattcGCCACCatggattccaaacagcagtgt-3ꢀ, contains an EcoRI site, a
Kozak sequence and a start codon, which are shown in italic,
capital and underlined letters, respectively. The antisense primer,
5ꢀ-ccggatccttaatattcatctgaatatggat-3ꢀ, is complementary to the bac-
terial expression vector containing a BamHI site, which is shown in
italic letters. After the sequences of the PCR products were verified,
they were subcloned at the EcoRI and BamHI sites of the pGW1
expression vector. Using a Lipofectamine 2000 Reagent (Invitro-
gen), the cells were transfected with the expression plasmids. The
transfected cells were maintained in the medium containing 2%
fetal bovine serum for 24 h, and then used for experiments. The
metabolism of GGOH and FOH were initiated by the addition of
1 ␮M [14C]GGOH and 20 ␮M [14C]FOH (each 1100 Bq), respectively,
to the medium of 90% confluent cells. A portion of the medium was
taken at different times, and finally the cells were collected and
homogenized. GGOH, FOH and their metabolites in the media and
cell homogenates were extracted into ethyl acetate, analyzed by
TLC, and their radioactivities were determined as described previ-
ously [24].
2.7. Mass spectrometry
The identity of the purified enzyme with known ADH or AKR
activities was examined by the mass spectrometry method. The
purified protein (0.1 mg) was digested by lysylendopeptidase or
trypsin, and then the digest was analyzed using a Bruker-Franzen
Mass Spectrometry System Ultraflex TOF/TOF with a saturated
␣-cyano-4-hydroxycinnamic acid matrix as described previously
[32].
3. Results and discussion
3.1. Intracellular distribution of oxidoreductases for isoprenoid
alcohols and aldehydes
When the intracellular distribution of the activities of the
NAD+-linked dehydrogenases (FOH-DH, GGOH-DH, FAL-DH and
GGAL-DH) and NADPH-linked reductases (FAL-R and GGAL-R) were
examined, more than 90% of the total activities of both FOH-DH
2.8. RT-PCR analysis
The total RNA samples were prepared from rat tissues and cul-
tured cells, and RT-PCR was carried out using Taq DNA polymerase

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S. Endo et al. / Chemico-Biological Interactions 191 (2011) 261–268
Table 1
Table 2
Activities of FOH-DH, GGOH-DH, FAL-DH, GGAL-DH, FAL-R and GGAL-R in the sub-
cellular fractions of rat liver, stomach and lung.
Comparison of properties among partially purified FOH-DHs and GGOH-DHs from
rat tissues and recombinant ADH1 and ADH7.
Enzyme
Specific activity (mU/mg) a
Enzyme partially purified from
Recombinant
Liver
Stomach
Lung
2.9 0.3
0.26 0.04
0.46 0.06
0.10 0.01
4.0 0.8
Liver
Stomach
Lung
Colon
ADH1
ADH7
Cytosolic FOH-DH
Cytosolic GGOH-DH
Microsomal FAL-DH
Microsomal GGAL-DH
Cytosolic FAL-R
18
11
11
3.2 1.1
8.1 0.5
1.3 0.4
4
3
2
5.4 2.2
0.74 0.28
0.84 0.26
0.26 0.06
2.7 0.8
Activity (U/mg) a for
Ethanol
FOH
0.31
0.29
0.20
0.070
0.032
0.006
0.014
0.012
0.003
0.067
0.054
0.022
0.73
0.68
0.45
62
43
3.3
GGOH
Km values (␮M) b for
Ethanol
Cytosolic GGAL-R
1.1 0.2
2.7 0.3
570
0.7
0.4
3400
29
0.8
2900
23
1.1
910
1.8
0.4
360
0.8
0.2
3700
26
0.9
FOH
GGOH
a
Specific activities of FOH-DH, GGOH-DH, FAL-DH, GGAL-DH, FAL-R and GGAL-
R were determined using 0.1 mM FOH, 50 ␮M GGOH, 50 ␮M FAL, 20 ␮M GGAL,
50 ␮M FAL and 20 ␮M GGAL, respectively, as the substrates at pH 7.4. The values are
means S.D. of three rats.
IC50 (␮M) c for
4-Methylpyrazole
1,10-Phenanthroline
13-cis-Retinoic acid
2.8
30
12
(36%)
35
12
(17%)
37
24
7.6
36
7.7
5.5
21
15
(25%)
20
15
a
Dehydrogenase activities for 40 mM ethanol, 0.1 mM FOH and 50 ␮M GGOH
and GGOH-DH were observed in the cytosolic fractions of the
homogenates of rat liver, stomach and lung. In contrast, FAL-DH
and GGAL-DH activities were detected only in the microsomal frac-
tions, and FAL-R and GGAL-R activities were recovered only in the
cytosolic fractions of the tissue homogenates. The products in the
oxidation of FOH, GGOH, FAL and GGAL by the subcellular fractions
were identified as FAL, GGAL, FA and GGA, respectively, by TLC, in
which their Rf values were identical to the respective standards.
Similarly, those in the reduction of FAL and GGAL by the cytosolic
fractions were identified as FOH and GGOH, respectively, by TLC
analysis. The representative TLC chromatograms of the products
formed by the incubation of the substrates with the hepatic sub-
cellular fractions are shown in Supplementary Fig. S1. The specific
activities of each enzyme in the cytosolic and microsomal fractions
of the tissue homogenates are shown in Table 1. Compared to the
extra-hepatic tissues, the liver showed high specific activities of
the four enzymes, except that the cytosolic GGAL-R activity was
high in the lung. In addition, the activity ratio of cytosolic FOH-DH
to GGOH-DH in the liver was 1.8, which was lower than those in
the stomach and lung (7 and 11, respectively). The activity ratio of
cytosolic FAL-R to GGAL-R in the liver was 6, which is in turn higher
than those (approximately 1.5) in the stomach and lung. These dif-
ferences suggest a possibility that the enzymes responsible for the
dehydrogenation of FOH and GGOH and for the reduction of FAL
and GGAL are different at least between the liver and the other
two tissues. In contrast, the activity ratios of microsomal FAL-DH
to GGAL-DH of the three tissues were constant.
were determined in 0.1 M potassium phosphate, pH 7.4, containing 2 mM NAD+.
b
The Km value represents the mean of three determinations in the above buffer.
The IC50 value for the FOH-DH activity was determined and represents the mean
c
of three determinations. The values in the parentheses are the inhibition % by 1 mM
inhibitor.
inhibitor sensitivity of the present rat microsomal FAL-DH are sim-
ilar to those reported for rat microsomal ALDH3A2 [33,35], which
is ubiquitously expressed in rat tissues [37]. The expression of the
mRNA for ALDH3A2 in rat liver, stomach, lung and colon was con-
firmed by RT-PCR (data not shown). Thus, ALDH3A2 may be the
enzyme that catalyzes the oxidation of FAL and GGAL into FA and
GGA, respectively.
3.3. Properties of cytosolic FOH-DH and GGOH-DH
In order to examine whether the different enzymes that are
responsible for the oxidation of FOH and GGOH depend on rat
tissues, we partially purified the cytosolic enzymes in the liver,
stomach, lung and colon by gel-filtration on the Sephadex G-100
column (Supplementary Fig. S2). In all analyses of the four tis-
sues, the FOH-DH and GGOH-DH activities were eluted at the same
high-MW position (around 80 kDa). The elution patterns were also
identical to that of the ethanol dehydrogenase activity, suggesting
that the activities of FOH-DH and GGOH-DH are attributed to ADH.
As ADH is rich in rat liver, the hepatic high-MW enzyme showed the
highest specific activities towards FOH and GGOH (Table 2). How-
ever, the activity ratios of FOH-DH to GGOH-DH in the high-MW
enzymes were different among the four tissues. The ratio was high
for the liver and colon enzymes, whereas it was low for the stomach
and lung enzymes. In addition, the liver and colon enzymes showed
lower Km values for ethanol and FOH than the gastric and pul-
monary enzymes. Furthermore, a similar difference in sensitivity
to 4-methylpyrazole, an ADH inhibitor [38], was observed among
the tissue enzymes, although all the tissue enzymes were inhibited
to a similar extent by other ADH inhibitors, 1,10-phenanthroline
and 13-cis-retinoic acid [39].
Three ADH isoenzymes, ADH1, ADH5 and ADH7, have been
purified from rat tissues, and differ in their substrate specificity
and tissue distribution [38–40]. Among the isoenzymes, ADH1 and
ADH7 are highly expressed in the liver and stomach, respectively,
and oxidize both ethanol and long-chain aliphatic alcohols. There-
fore, we prepared recombinant ADH1 and ADH7, and compared
their properties with those of the high-MW FOH-DH and GGOH-
DH partially purified from rat tissues (Table 2). With respect to
the activity ratios among ethanol, FOH and GGOH, Km values for
the substrates and sensitivity to 4-methylpyrazole, ADH1 resem-
bles the enzymes in the liver and colon, and ADH7 is similar
to the enzymes in the stomach and lung. In addition, the lung
and stomach enzymes were almost completely immunoprecipi-
3.2. Properties of microsomal FAL-DH and GGAL-DH
More than 90% of the NAD+-linked FAL-DH and GGAL-DH activi-
ties in the microsomal fractions of rat liver, stomach and lung were
solubilized by the treatment with 1% Triton X-100 at 4 ◦C for 1 h.
Using the solubilized preparation of the hepatic microsomes, the
kinetic constants for the substrates, coenzyme requirement and
inhibitor sensitivity were examined. The Km and Vmax values for
FAL were 2.0 ␮M and 12 mU/mg, respectively, and the respective
values for GGAL were 0.7 ␮M and 4.2 mU/mg. When 2 mM NADP+
was used as the coenzyme, low FAL-DH activity of 2.5 mU/mg was
observed. The NAD+-linked FAL-DH activity was completely inac-
tivated by incubation with 10 ␮M p-chloromercuribenzoate for
5 min at 25 ◦C, and inhibited by chloral hydrate (IC50 = 28 3 ␮M)
and 4-hydroxyacetophenone (IC50 = 13 1 ␮M), which are known
inhibitors of rat and human microsomal ALDHs [34,35].
An ALDH isoenzyme and its cDNA have been isolated from
rat liver microsomes [36,37], and this is the only microsomal
ALDH isoenzyme (ALDH3A2) found in the rat genome database
(http://rgd.mcw.edu). The isoenzyme is also called fatty aldehyde
dehydrogenase because of its high affinity towards aliphatic alde-
hydes of 7–9 carbon chains [36]. The dual coenzyme specificity and

S. Endo et al. / Chemico-Biological Interactions 191 (2011) 261–268
265
Fig. 2. Identification of ADH7 as a major enzyme exhibiting FOH-DH and GGOH-DH activities in rat lung, stomach and gastric mucosa RGM1 cells. (A) Immunoprecipitation
of FOH-DH activity by the anti-ADH7 antibody. The antibody was incubated with recombinant enzymes (dotted lines), ADH1 (ꢀ) and ADH7 (᭹), and the high-MW enzymes
(solid lines) purified from rat liver (ꢀ), colon (ꢁ), lung (ꢁ) and stomach (ꢀ) at 4 ◦C for 12 h. After the immunocomplexes were removed by centrifugation, the activity in the
supernatant was determined and is expressed as the percentage of that incubated without the antibody. (B) TLC chromatogram of the metabolites of GGOH in the medium
(m) and RGM1 cells (c), which were incubated with 1 ␮M [14C] GGOH for 0 and 8 h. The positions of authentic samples are shown with arrow heads in the right side. Unknown
components present at 0-h culture are shown with asterisks. (C) Effect of transfection of the anti-ADH7 antibody on metabolism of GGOH in RGM1 cells. The cells were
cultured 24 h after transfection with the antibody, and then incubated with 1 ␮M [14C] GGOH. A portion of the medium was taken at indicated times for determination of
GGOH (ꢁ, ᭹) and GGA (ꢀ, ꢁ) in the non-transfected (open symbols and solid lines) and transfected cells (closed symbols and dotted lines). Relative radioactivities of GGOH
and GGA were calculated as described in Section 2.
tated by the anti-ADH7 antibodies that did not precipitate the
hepatic and colonic enzymes and ADH1 (Fig. 2A). When the liver
and stomach enzymes were purified to homogeneity and their
lysylendopeptidase-digested peptides were analyzed by the mass
spectrometry method, the sequence (composed of a total 106
residues) of the liver enzyme and that (composed of a total 225
residues) of the stomach enzyme were identical to those deduced
from the cDNAs for ADH1 and ADH7, respectively. These results
clearly indicate that the oxidation of FOH and GGOH in the rat
tissues is predominantly catalyzed by the two tissue-specific ADH
isoenzymes. The kcat/Km values for GGOH of ADH1 and ADH7 were
90 and 150 min−1 ␮M−1, respectively, which are higher than the
that is known as the only rat enzyme being able to reduce FAL
[21]. AKR1C15 reduced GGAL more efficiently than FAL: The Km
and kcat/Km values for GGAL were of 0.2 ␮M and 6.0 min−1 ␮M−1
respectively, while the respective values for FAL are reported to
be 5.6 ␮M and 2.1 min−1 ␮M−1. When the Km values for the iso-
prenyl aldehydes were compared among the low-MW reductases
from the rat four tissues, the liver enzyme showed higher values
for FAL and GGAL (26 and 2.1 ␮M, respectively) than the enzymes
from the stomach, lung and colon. The Km values of the extra-
hepatic enzymes for FAL and GGAL are 6–8 ␮M and 0.2–0.5 ␮M,
respectively, which are similar to those of AKR1C15. In addi-
tion, the liver enzyme differed from the other tissue enzymes
in sensitivity to potent AKR1C15 inhibitors (3ꢀ,3ꢀꢀ,5ꢀ,5ꢀꢀ-tetraiodo-
phenolphthalein and 3ꢀ,3ꢀꢀ,5ꢀ,5ꢀꢀ-tetrabromophenolphthalein) [21]:
The two inhibitors (1 ␮M) showed less than 20% inhibition for the
liver enzyme activity, but inhibited more than 80% of the activi-
ties of the stomach, lung and colon. The results suggest that the
low-MW FAL-R and GGAL-R in the three extra-hepatic tissues are
identical to AKR1C15, but other reductase(s) for FAL and GGAL exist
in the liver. This was further supported by immunoprecipitation
using the anti-AKR1C15 antibody which monospecifically reacts to
the antigen [21]. The FAL-R activities from the stomach, lung and
colon were decreased by the antibody in a dose-dependent manner,
but only 20% of the enzyme activity of the liver was precipitated at
high concentrations of the antibody (Fig. 3A). Thus, it is concluded
that in the stomach, lung and colon AKR1C15 acts as a predominant
reductase for FAL and GGAL.
The above results suggest that in the liver other reductase(s) for
the isoprenyl aldehydes may be expressed more than AKR1C15.
To identify the major form of the hepatic low-MW reductases
for FAL and GGAL, we purified the enzyme to homogeneity from
the low-MW fraction obtained by the gel-filtration. The purified
enzyme reduced both FAL and GGAL, and showed higher Km for FAL
and GGAL (30 and 3.9 ␮M, respectively) and lower kcat/Km values
(0.2 and 0.3 min−1 ␮M−1, respectively) than AKR1C15. This enzyme
also exhibited high reductase activity towards 3-ketosteroids: For
example, the Km and kcat values for 5␤-androstane-3,17-dione
were 1.6 ␮M and 25 min−1, respectively. In addition, more than
90% of the FAL-R activity of the purified enzyme was inhib-
ited by 5 ␮M medroxyprogesterone acetate, 5 ␮M indomethacin
and 50 ␮M betamethasone, which are inhibitors of AKR1C9, i.e.,
rat 3␣-hydroxysteroid dehydrogenase [41,42]. Furthermore, the
values of the two isoenzymes for FOH (34 and 67 min−1 ␮M−1
,
respectively). Although the FOH-DH activity of horse and human
ADHs have been reported [13,14], this study demonstrates for the
first time that ADH efficiently oxidizes GGOH.
To further confirm the oxidation of GGOH into GGAL by the
tissue-specific ADH isoenzymes and involvement of the micro-
somal ALDH3A2 in the subsequent metabolism into GGA, we
investigated the metabolism of GGOH in the gastric mucosa RGM1
cells, in which the expression of the mRNAs for ADH7 and ALDH3A2,
but not that for ADH1, were confirmed by RT-PCR (data not shown).
GGOH was metabolized into GGA (Fig. 2B), which accumulated in
the medium proportionally with the decrease in GGOH (Fig. 2C),
indicating that the metabolized GGA is excreted from the cells.
While unknown metabolites with high Rf values were observed in
the cells, GGAL was not detected in both medium and cells prob-
ably because of its rapid metabolism into GGA by ALDH3A2. The
metabolic rate from GGOH into GGA was significantly decreased
by introducing the anti-ADH7-antibodies into the cells (Fig. 2C).
These results indicate that GGOH is mainly metabolized into GGA
via GGAL by ADH7 and ALDH3A2 in the cells, and also support the
predominant role of ADH7 in the first step of the GGOH catabolism
in rat stomach.
3.4. Properties of cytosolic FAL-R and GGAL-R
In the gel-filtration analysis of the cytosolic proteins in the
liver, stomach, lung and colon, the FAL-R and GGAL-R activities
were co-eluted at the low-MW fraction, which is distinct from
the high-MW FOH-DH activity. The molecular masses of the tis-
sue FAL-R and GGAL-R are similar to that of AKR1C15 (36 kDa)

266
S. Endo et al. / Chemico-Biological Interactions 191 (2011) 261–268
which were employed because the expression plasmid could not
be transfected into rat RGM1 cells. On Western blot analysis using
the anti-AKR1C15 antibody, a 36-kDa immunoreactive band was
detected only in the transfected HeLa cells, which showed 5-fold
higher FAL-R activity (1.9 mU/mg) than the control cells trans-
fected with the vector alone. FOH was rapidly converted into FA
in the control cells, in which FA accumulated in the medium and
FAL was hardly observed, similarly to the metabolism of GGOH in
RGM1 cells. Compared to the control cells, the metabolic rate from
FOH to FA was greatly decreased in the AKR1C15-overexpressed
cells (Fig. 3B), indicating that AKR1C15 reduces low concentra-
tions of FAL produced from the oxidation of the added FOH. This
supports the proposed role of the reductase in controlling the cel-
lular concentrations of FOH and GGOH, which are utilized for sterol
synthesis and protein prenylation, and exhibit multiple biological
actions [2,3,6–10].
3.5. Human reductases for FAL and GGAL
Considering the role of rat AKRs in regulating the metabolic rates
from biologically active FOH and GGOH to their carboxylic acids, it
is particularly interesting to identify human enzymes that function
as FAL-R and GGAL-R. Human enzymes in the AKR superfamily dif-
fer from rodent enzymes in molecular species and properties [44],
and four human AKRs (1C1–1C4) belong to the AKR1C subfamily.
The kinetic constants of the four AKRs for FAL and GGAL were deter-
mined and compared with those of human AKR1B10 and AKR1B1,
which are reported to reduce the two isoprenyl aldehydes [24]
(Table 3). FAL was most efficiently reduced by AKR1B10, followed
by AKR1C3 and AKR1C2. AKR1C3 and AKR1B10 were also excellent
reductases towards GGAL. In particular, the Km value of AKR1C3 for
GGAL is lower than those for previously known substrates (steroids
and prostaglandins), and its kcat/Km value is 12-fold higher than that
for prostaglandin D2 which has been recognized as the best endoge-
nous substrate of this enzyme [31,44]. It should be noted that FAL
and GGAL were hardly reduced by human carbonyl reductase 1,
dicarbonyl-xylulose reductase and dehydrogenase/reductase (SDR
family) member 4, which exhibit broad substrate specificity for
carbonyl compounds and belong to the short-chain dehydroge-
nase/reductase superfamily [44].
Since AKR1C3 efficiently reduced FAL and GGAL in vitro, its con-
tribution to cellular metabolism of FAL was examined using MCF7
cells instead of HeLa cells (Fig. 4). RT-PCR analysis revealed that
MCF7 cells expressed the mRNAs for AKR1C1, AKR1C2 and AKR1C3
more highly than HeLa cells, although the two cell lines showed no
significant expression of the mRNAs for AKR1B10 and AKR1C4 (data
not shown). The metabolic rate from FOH to FA (i.e., the amount of
FA formed) in MCF7 cells was low compared to that in HeLa cells
(i.e., the FA amount in the 6 h-incubated control cells in Fig. 3B).
This suggests that AKR1C1, AKR1C2 and/or AKR1C3 act as FAL-R
and reduce FAL back to FOH. To test this possibility and identify
the major FAL-R species, we examined the effects of inhibitors of
human AKRs on the cellular metabolism of FOH. Since nonsteroidal
anti-inflammatory drugs have been reported to inhibit human
AKRs (1B10, 1C1, 1C2 and 1C3) [45,46,47], we first compared
the inhibitory effects of the drugs on the AKRs in vitro. AKR1C3
was potently inhibited by several nonsteroidal anti-inflammatory
drugs (Table 4), of which tolfenamic acid and R-flurbiprofen were
most selective to AKR1C3 and showed competitive inhibition with
respect to the substrate S-(+)-1,2,3,4-tetrahydro-1-naphthol (Ki
values were 8 and 610 nM, respectively). Tolfenamic acid and R-
flurbiprofen increased the formation of FA from FOH in the MCF7
cells in a dose-dependent manner (Fig. 4). In contrast, no sig-
nificant changes in the cellular FOH metabolism were observed
by the addition of (Z)-2-(4-methoxyphenylimino)-7-hydroxy-N-
(pyridin-2-yl)-2H-chromene-3-carboxamide (a potent inhibitor of
Fig. 3. Identification of AKR1C15 as a major enzyme exhibiting FAL-R activity in
rat tissues. (A) Immunoprecipitation of FAL-R activity by the anti-AKR1C15 anti-
body. The immunoprecipitation was carried out as described in Fig. 2A. Enzymes
are AKR1C15 (ꢁ- - -ꢁ) and the low-MW enzymes (solid lines) purified from rat
liver (ꢁ), lung (ꢁ), colon (ꢀ), and stomach (᭹). (B) Effect of the overexpression of
AKR1C15 on metabolism of FOH in HeLa cells. The cells were cultured 24 h after
transfection with the expression plasmids harboring the cDNA for AKR1C15, and
then incubated with 20 ␮M [14C] FOH. A portion of the medium was taken at indi-
cated times, and the radioactivities of FOH (ꢁ, ᭹) and FA (ꢀ, ꢁ) in the media of the
control (open symbols and solid lines) and AKR1C15-overexpressed cells (closed
symbols and dotted lines) were determined as described in Section 2.
sequences (composed of a total 94 residues) of nine tryptic pep-
tides derived from the purified enzyme were identical to those
deduced from the cDNA for AKR1C9. Indeed, recombinant AKR1C9
reduced FAL and GGAL and its kinetic constants for the substrates
were almost the same as those of the purified liver enzyme. The
results clearly indicate that the hepatic FAL-R and GGAL-R activi-
ties other than those by AKR1C15 are mostly due to AKR1C9, which
is highly expressed in rat liver [43].
The above results demonstrate the presence of the reductases
for FAL and GGAL in rat tissues, suggesting that the enzymes play
a role in regulation of the metabolic rates from FOH and GGOH to
their carboxylic acids by reducing FAL and GGAL back into FOH and
GGOH, respectively. In spite of the lower kcat values for FAL and
GGAL of AKR1C15 than those for FOH and GGOH of ADH isoen-
zymes, the gel-filtration analysis indicated that AKR1C15 is highly
expressed in the extra-hepatic tissues. AKR1C15 is a more effi-
cient isoprenyl aldehyde reductase than AKR1C9 in vitro, and is
expressed in many rat tissues including vascular endothelial cells
[21]. Therefore, we further examined the role of AKR1C15 in the
metabolism of FOH using the AKR1C15-overexpressing HeLa cells,

S. Endo et al. / Chemico-Biological Interactions 191 (2011) 261–268
267
Table 3
Kinetic constants for FAL and GGAL of human AKRs.
AKR
FALa
GGALa
Km (␮M)
kcat (min−1
)
kcat/Km (min−1 ␮M−1
)
Km (␮M)
kcat (min−1
)
kcat/Km (min−1 ␮M−1
)
1A1
1B1
1B10
1C1
1C2
1C3
1C4
60
37
13
27
23
1.7
1.8
2.7
1.1
0.22
0.73
9.1
0.55
1.6
(0.8) b
(0.3) b
7.5
2.5
3.1
1.1
2.6
4.7
0.9
8.3
(0.3) b
(0.4) b
3.6
1.1
0.23
0.3
1.1
12
0.80
0.88
a
The kinetic constants for FAL and GGAL were determined in 0.1 M potassium phosphate, pH 7.4, containing a saturated concentration (0.1 mM) of NADPH.
The value in parenthesis is calculated from the specific activity with 10 ␮M GGAL.
b
Table 4
Inhibitory effects of nonsteroidal anti-inflammatory drugs on human AKRs.
Drug
IC50 value (␮M) a
AKR 1A1
AKR 1B1
AKR 1B10
AKR 1C1
AKR 1C2
AKR 1C3
AKR 1C4
Tolfenamic acid
Mefenamic acid
Meclofenamic acid
R-flurbiprofen
30
28
23
>100 b
77
21
35
58
>100 b
1.6
12
87
87
0.71
2.2
0.57
2.6
0.017
0.11
0.43
1.5
53
97
1.1
2.2
>100 b
Act c
Act c
>50 b
>50 b
>50 a
>50 a
>50 a
>50 a
S-flurbiprofen
5.9
a
The values were determined in the reduction of 10 mM d-glucuronate (for AKR1A1) and 0.2 mM pyridine-3-aldehyde (for AKR1B1 and AKR1B10) in 0.1 M potassium
phosphate, pH 7.4, containing 0.1 mM NADPH. The IC50 values for the AKR1C isoforms in the oxidative direction were assayed using 0.1 mM (for AKR1C1) and 1.0 mM
S-(+)-1,2,3,4-tetrahydro-1-naphthol (for the other isoforms) as the substrates in the phosphate buffer containing 0.25 mM NADP+.
b
Less than 30% inhibition at the indicated concentration.
Activation.
c
both AKR1B10 and AKR1B1) [48] and 3-bromo-5-phenylsalicylic
acid that is a more potent inhibitior of AKR1C1 than AKR1C2 [23].
The data reveal that AKR1C3 is the major FAL-R in MCF7 cells, and
significantly affects the catabolic rate from FOH to FA.
In human cells that highly expresses AKR1C3 like MCF7 cells,
the enzyme may also act as GGAL-R because of its high catalytic
efficiency for GGAL. In the metabolism of GGOH, the reversible
oxidation of GGOH into GGAL by ADH and AKRs may also be
the limiting step in the pathway for the synthesis of GGA and
2,3-dihydrogeranylgeranoic acid, which exhibit biological actions
on several cultured cells [16,17,18]. AKR1C3 is suggested to be
involved in proliferation of breast and prostate cancers through
the metabolism of androgen and prostaglandins [49]. AKR1B10,
another human enzyme that efficiently reduces FAL and GGAL,
is elevated in cancer lesions such as liver and lung cancers, and
suggested to be involved in cancer cell proliferation through the
metabolism of retinoids and fatty acids [50]. Considering the anti-
cancer action of GGA against human hepatic cancer cells [16],
elevated AKR1C3 or AKR1B10 might be responsible for proliferation
of the cancer cells by preventing the production of anti-cancerous
GGA, as well as by providing GGOH which is the substrate in
prenylation of small G-proteins. Further studies in our laborato-
ries are underway to elucidate the human enzymes in the proposed
reversible oxidation of the isoprenols and their regulatory functions
of human cancerous cells.
4. Conflicts of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was supported by Grant-in-Aids for young scientists
and scientific research from the Japan Society for the Promotion
of Science, and by a grant for encouragement of young scientists
from Gifu Pharmaceutical University. SE was supported by USPHS
NIH grant R13-AA019612 to present this work at the 15th Interna-
tional Meeting on Enzymology and Molecular Biology of Carbonyl
Metabolism in Lexington, KY USA.
Fig. 4. Inhibition of FAL reduction in MCF7 cells by selective AKR inhibitors. The cells
were incubated for 6 h with 20 ␮M [14C] FOH in the absence or presence of inhibitors
for AKR1C3 (Tol: tolfenamic acid and R-Flu: R-flurbiprofen), AKR1C1 (BPS: 3-
bromo-5-phenylsalicylic acid) and AKR1B10 (C1: (Z)-2-(4-methoxyphenylimino)-
7-hydroxy-N-(pyridin-2-yl)-2H-chromene-3-carboxamide). The radioactivities of
FOH and FA in the media of duplicate experiments were measured, and are expressed
as the mean percentages, relative to their sum.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at 10.1016/j.cbi.2010.12.017.

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S. Endo et al. / Chemico-Biological Interactions 191 (2011) 261–268
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