Chronic Ethanol Consumption Alters All-Trans-Retinoic
Acid Concentration and Expression of Their Receptors on
the Prostate: A Possible Link Between Alcoholism and
Beatriz A. F. Fontanelli, Luiz G. A. Chuffa, Giovana R. Teixeira, Joa ˜o P. A. Amorim, Leonardo
O. Mendes, Patricia F. F. Pinheiro, Cilmery S. Kurokawa, Se ´rgio Pereira, Wagner J. Fa ´varo,
Ota ´vio A. Martins, Wı ´lson Mello Ju ´nior, Marcelo Martinez, Antonio Rugolo Ju ´nior, and
Francisco E. Martinez
and ATRA are essential for cell proliferation, differentiation, and maintenance of prostate homeostasis.
inoic acid receptors (RARs) contribute to benign prostate hyperplasia and prostate cancer. This study
aimed to evaluate whether EtOH consumption is able to alter retinol and ATRA levels in the plasma
Methods: All animals were divided into 4 groups (n = 10/group). UChA: rats fed 10% (v/v) EtOH
ad libitum; UChACo: EtOH-naı¨ve rats without access to EtOH; UChB: rats fed 10% (v/v) EtOH ad
libitum; UChBCo: EtOH-naı¨ve rats without access to EtOH. Animals were euthanized by decapitation
after 60 days ofEtOH consumption for high-performance liquid chromatographyand light microscopy
Results: EtOH reduced plasma retinol concentration in both UChA and UChB groups, while the
retinol concentration was not significantly different in prostate tissue. Conversely, plasma and prostate
ATRA levels increased in UChB group compared with controls, beyond the up-regulation of RARb
and -c in dorsal prostate lobe. Additionally, no alteration was found in cell proliferation and apoptosis
index involving dorsal and lateral prostate lobe.
Conclusions: We conclude that EtOH alters the plasma retinol concentrations proportionally to the
amount of EtOH consumed. Moreover, high EtOH consumption increases the concentration of ATRA
in plasma/prostate tissue and especially induces the RARb and RARc in the dorsal prostate lobe.
EtOH consumption and increased ATRA levels were not associated with cell proliferation and apopto-
sis in the prostate.
Key Words: Retinol, All-Trans-Retinoic Acid, Retinoic Acid Receptor, Prostate, Ethanol.
reproductive tract (Chuffa et al., 2009; Martinez et al.,
2000, 2001a,b). It is well documented that EtOH con-
sumption leads to atrophy of the prostate epithelium
HRONIC ETHANOL (ETOH) consumption is asso-
ciated with morpho-physiological changes in the
(Martinez et al., 2001a), disruption of stromal–epithelial
2007), and increased inflammatory activity of mast cells
(Mendes et al., 2011). Moreover, epidemiological studies
have shown the relationship between chronic and excessive
developing prostate cancer (Hirayama, 1992; Sharpe and
EtOH exposure alters the metabolism and concentration
of retinol (vitamin A) and its metabolite, retinoic acid, in the
plasma, liver, hippocampus, and testis (Kane et al., 2010;
Leo and Lieber, 1982), but so far, none has evaluated the
effects of EtOH upon physiological concentration of retinol
and retinoic acid in the prostate. EtOH also decreases the
concentration of retinoic acid in the liver, thus increasing the
proliferation of hepatocytes which contributes to the EtOH-
related carcinogenesis. Conversely, elevation in retinoic acid
levels seems to cause cytotoxicity, especially for tissues that
require retinoic acid during its development (Seitz and
Stickel, 2007; Wang, 2005).
neoplasia (Ca ˆ ndidoet al.,
an accentuatedrisk of
From the Department of Anatomy (BAFF, LGAC, GRT, JPAA,
LOM, PFFP, SP, WJF, OAM, WMJ, FEM), IBB/UNESP – Univ Es-
tadual Paulista, Botucatu, SP, Brazil; Structural and Cell Biology Pro-
gram (BAFF, LGAC, JPAA, LOM), UNICAMP, Campinas, SP,
Brazil; Department of Pediatrics (CSK, ARJ), FMB/UNESP – Univ Es-
tadual Paulista, Botucatu, SP, Brazil; and Department of Morphology
and Pathology (MM), UFSCar – Federal University of Sa˜o Carlos, Sa˜o
Carlos, SP, Brazil.
Received for publication July 22, 2011; accepted March 27, 2012.
Reprint requests: Francisco E. Martinez, Department of Anatomy,
Institute of Bioscience, UNESP – Univ Estadual Paulista, PO Box 510,
Rubia˜o Ju ´nior, s/n, Botucatu, SP 18618-970, Brazil; Tel.: +55-(14)-
3811-6040; Fax: +55-(14)-3811-6361; E-mail: email@example.com
Copyright © 2012 by the Research Society on Alcoholism.
Alcohol Clin Exp Res,Vol **, No *, 2012: pp 1–81
ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH
Vol. **, No. *
Retinoic acid is fundamental to development, prolifera-
tion, differentiation, and maintenance of prostate homeosta-
sis (Lasnitzki and Goodman, 1974; Peehl et al., 1993; Prins
and Putz, 2008; Vezina et al., 2008). The oxidation of retinol
firstly generates 2 acids, all-trans-retinoic acid (ATRA) and
9-cis-retinoic acid; however, ATRA is the most abundant
and the only detectable isoform at normal concentrations of
retinol (Dong and Zile, 1995; Heyman et al., 1992). It should
be noted that cellular signaling such as proliferation, differ-
entiation, and apoptosis are controlled by ATRA binding to
its receptors (RARa, RARb, and RARc), in a concentra-
tion-dependent manner (De Luca, 1991; Fields et al., 2007;
Kastner et al., 1997). Furthermore, changes in retinol and
retinoic acid concentrations as well as in RARs expression
have been described during benign prostatic hyperplasia
(BPH) and prostate cancer, suggesting that this pathway
plays an important role in developing prostate diseases (Pas-
quali et al., 1996, 1999; Richter et al., 2002). In view of the
above-mentioned reports, the investigation of EtOH con-
sumption upon the concentration and signalization of retinol
and ATRA focusing the prostate tissue would bring new
insights into the harmful effects of EtOH.
Therefore, this study aimed to evaluate whether EtOH
consumption is able to alter retinol and ATRA levels in the
plasma and prostate tissue as wellas the expression of RARs,
cell proliferation, and apoptosis index.
MATERIALS AND METHODS
Animals and Experimental Groups
Forty UCh rats were selected for this study. Twenty adult male
rats of each variety, UChA and UChB, weighing between 280 and
330 g (~90 days old), were obtained from the Department of Anat-
omy, Bioscience Institute/Campus of Botucatu (IBB), UNESP-
Univ Estadual Paulista. These varieties are derived from Wistar rats
and they have been selectively bred at University of Chile for low
consumption preference (UChA—0.5 to 1.9 g/kg body weight
(BW)/d) and high consumption preference (UChB—2.0 to 6.0 g/kg
BW/d) of EtOH solution 10% over several decades (Mardones and
Segovia-Riquelmi, 1983; Quintanilla et al., 2006). UChA and
UChB rats were subdivided into 2 experimental groups (n = 10⁄
group) and respective controls. UChA group: rats fed 10% (v/v)
EtOH ad libitum (free choice for water or EtOH); UChACo group:
EtOH-naı¨ve rats without access to EtOH, used as a control group;
UChB group: rats fed 10% (v/v) EtOH ad libitum (free choice for
water or EtOH); UChBCo group: EtOH-naı¨ve rats without access
to EtOH, used as a control group. All animals were housed in indi-
vidual cages in a temperature- and humidity-controlled room under
a 12-hour light/dark cycle and had free access to filtered tap water
and standard rodent chow Nuvital®(Nuvilab CR-1, Colombo, PR,
Brazil). Experimental protocols followed the Ethical Principles in
Animal Research adopted by the Brazilian College of Animal
Experimentation and were approved by Ethics Committee on Ani-
mal Experimentation (protocol no. 116/09).
EtOH, the Amount of Energy from EtOH and Food Consumption
EtOH ingestion was measured using a marked test tube for assess
the profile of EtOH consumption of UCh variety during experimen-
tal period. The chow consumption was evaluated using a analytical
balance (Ohaus TravelerTM, Mexico, D.F, MEXICO, MX) for
measure the amount of retinol consumed based on quantity of
vitamin A (25,500 UI) of chow Nuvilab CR-1 (1 UI = 0.3 lg all-
trans-retinol) (Institute of Medicine, 2000). EtOH and chow con-
sumptions were measured once a week. The energy from EtOH was
calculated by EtOH ingested amount by the amount of calories
from EtOH (1 g EtOH = 7.1 kcal).
Analysis of the Retinol and ATRA Concentration in Plasma and
Prostate Tissue by High-Performance Liquid Chromatography
Rats were sacrificed by decapitation after 8 weeks of experimen-
tation. The blood was collected and centrifuged at 6989g for
15 minutes and the plasma stored at ?20°C. The right dorsolateral
lobe of prostate was removed and weighed using an analytical bal-
ance (OwaLabor, Oschatz, Germany) and then, frozen in liquid
nitrogen and stored at ?80°C. Samples of 250 mg prostate tissue
was homogenized in purified water (1:1 [w/w]) (Millipore, Billerica,
MA) using a T-10 basic Ultra-Turrax homogenizer (Ika, Staufen,
Germany). Retinol was extracted from prostatic tissue and plasma
according to the method previously described by Arnaud and col-
leagues (1991), while ATRA extraction was based on the method
described by Kane and colleagues (2005). To ensure more efficiency
of extraction, each sample was extracted 3 times and the superna-
tant removed and evaporated using a centrifugal evaporator system
Speed Vac SC100 (Savant, Inc., Farmingdale, NY). All retinol and
ATRA residues were reconstituted with 100 ll acetonitrile/metha-
nol/dichloromethane (70:20:10 [v/v]) and 100 ll acetonitrile, respec-
tively. Thereafter, samples were individually injected into the high-
performance liquid chromatography (HPLC) system (Varian 9012/
9050). Each sample containing retinol was loaded onto an analytical
reverse-phase C18column, 5-lm particle size (Chrompack Varian,
Hesperia, CA) and eluted at a flow rate of 1.2 ml/min. Sample con-
taining ATRA was loaded onto an analytical reverse-phase, 3-lm
particle size (Supersil ODS, Belvedere, CA) and eluted at a flow rate
of 0.5 ml/min. The mobile phase used to separate the retinol and
ATRA was composed of the same solvents that were reconstituted.
Retinol and ATRA were detected at 325 and 340 nm, respectively,
and quantified by determining peak areas on high-performance
liquid chromatograms, calibrated against known amounts of stan-
dards. Retinyl palmitate and all-trans-acitretin was used as internal
control of retinol and ATRA, respectively, to determine the effi-
ciency of extraction. Efficiency extraction retinol was more than
90% and of ATRA more than 80%. As retinol and ATRA are sen-
sitive to light exposure, all experiments (collection, sample prepara-
tion as well as the HPLC analysis) were carried out under dim
yellow light (Schmidt et al., 2003).
Analysis of Retinoic Acid Receptors (RARa, RARb, and RARc) and
Cell Proliferation by Immunohistochemistry
The left dorsolateral lobe of prostate was dissected, separated
into dorsal and lateral prostate lobes, and then, fixed in 10% phos-
phate-buffered formalin (0.1 M, pH 7.2) and embedded in paraplas-
tic. Sections of 4-lm thick of the dorsal and lateral prostate lobes (5
samples/group) were placed on silane-coated slides. Following to
antigen retrieval, the exposure of RARs epitopes was performed
placing the slices in sodium citrate buffer, pH 6.0 and heated 3 times
for 5 minutes using a microwave, while protein Ki-67 was recovered
using a pressure cooker for 30 minutes. Primary antibodies anti-
RARa diluted at 1:50 (Santa Cruz Biotechnology, Inc., Santa Cruz,
CA); anti-RARb diluted 1:150 (Abcam, Inc., Cambridge, MA);
anti-RARc diluted 1:750 (Imuny Biotechnology, Campinas, SP,
Brasil) and anti-Ki67 diluted 1:100 (Novocastra, Inc., Newcastle
Upon Tine, UK) were applied to the sections and incubated in a
moist chamber at 4°C overnight. The primary antibody anti-RARa,
anti-RARb and anti-RARc were detected using a Polymer
conjugated to peroxidase (Novolink Polymer; Novocastra, Inc.).
FONTANELLI ET AL.
Antibody anti-ki67 was detected by secondary biotinylated anti-
body (Dako, Copenhagen, Denmark) followed by avidin–biotin–
peroxidase incubation (Vector Laboratory, Inc., Burlingame, CA).
All reactions were finally revealed with 3,3′ diaminobenzidine tetra-
hydrochloride chromogen (Sigma-Aldrich, St. Louis, MO) and sec-
tions were counterstained with Harris’s hematoxylin. Negative
controls were obtained without primary antibody. All sections were
examined under microscope Olympus BX-41 and images captured
by digital camera DP-12 (Olympus, Inc., Tokyo, Japan).
Detection of Apoptotic Cells by TUNEL Assay
Apoptotic cells were evaluated by using terminal deoxynucleotide
transferase-mediated deoxyuridine triphosphate nick end labeling
(TUNEL) assay. In Situ Cell Death Detection Kit (TdT-Fragel-Cal-
biochem, La Jolla, CA) was used for the detection of DNA frag-
mentation, following the manufacturer’s instructions. Slides were
counterstained with Harris’s hematoxylin. Negative controls were
not incubated with the TdT enzyme.
Two-way analysis of variance (ANOVA) was performed to deter-
mine the differences for quantitative HPLC, Ki-67, and TUNEL
among the groups followed by Tukey’s post hoc test. Statistical
analyses for RARa, RARb, and RARc proteins were performed by
a nonparametric Kruskal–Wallis test complemented by Dunn’s
multiple comparisons test. Statistical significance was set at
p < 0.05. The statistical software used was Graph Pad Instat version
3 (Graph Pad Software, San Diego, CA) and Sigma Plot version
11.0 (Systat Software, Inc., Chicago, IL) for graphic design.
EtOH, the Amount of Energy from EtOH and Retinol
EtOH consumption, the amount of energy from EtOH
and food-derived retinol intake were measured throughout
the experimental period (Table 1). The amount of retinol
consumed by both groups was not different (Table 1).
Concentration of Retinol and ATRA in the Plasma and
Ethanol consuming UChB rats had a reduced plasma reti-
nol concentration at 1.8 times compared with UChBCo
(p < 0.01), and similarly, the UChA rats also reduced plasma
retinol concentration at 1.3 times (p < 0.05) compared to
UChACo, but it was unchanged in the prostate (Fig. 1A,B).
Plasma retinol concentration in UChB-EtOH drinking rats
was about 2 times lower than UChA rats (p < 0.001). On the
other hand, plasma ATRA concentration increased approxi-
mately 2 times (p < 0.05) during high EtOH consumption,
while in the prostate, it was increased by 2.4 times (p < 0.05;
Fig. 2A). Conversely, low EtOH consumption did not signif-
icantly alter the ATRA concentration in both plasma and
prostate (Fig. 2B).
Analysis of Retinoic Acid Receptors (RARa, RARb, and
The RARa and RARb were located along dorsal and
lateral prostate lobes in all experimental groups, whereas
RARc was found only in the dorsal prostate lobes. Table 2
shows the percentage of RARa, RARb, and RARc reactivity
in the prostate among the groups. RARa was located in the
nucleus of basal and luminal cells of dorsal and lateral
prostate lobes. The analysis of RARa did not present any
differences for immunoreactivity (Figs 3A–D and 4A–D).
Table 1. Relative Consumption of Ethanol (EtOH) (g/kg/d), the Amount of
Energy from EtOH (kcal) and Food (g/kg/d) and Retinol Intake (lg/kg/d)
Liquid and Solid Diet
EtOH Energy from EtOH FoodRetinol
1.3 ± 0.11
4.0 ± 0.3
9.2 ± 0.8
28.4 ± 2.1
82.1 ± 1.2
83.0 ± 3.1
86.3 ± 5.8
73.3 ± 2.7
0.63 ± 0.05
0.63 ± 0.03
0.66 ± 0.14
0.57 ± 0.08
There was no significant difference among the groups (p > 0.05). Data
are expressed as mean ± SEM (n = 10/group). Two-way ANOVA comple-
mented by Tukey’s test.
Fig. 1. Quantification of endogenous retinol levels in plasma and pros-
tate. (A) UChB/UChBCo groups and (B) UChA/UChACo groups. Values
are expressed as mean ± SEM (n = 10/group). **p < 0.01 versus
UChBCo, *p < 0.05 versus UChACo.
ETHANOL ALTERS ATRA/RARs ON THE PROSTATE
The RARb was found in both nucleus and cytoplasm
of luminal and basal cells over the dorsal prostate lobe
(Fig. 3E–H); however, in the lateral prostate lobe, it was
only detected in the cytoplasm and nucleus of the basal cells
(Fig. 4E–H). In dorsal and lateral prostate lobes, the positiv-
ity for RARb was lower than RARa. The high EtOH con-
sumption (UChB) increased RARb in the dorsal prostate
lobe compared with the UChBCo (Table 2).
The RARc was mainly observed in the nucleus and cyto-
plasm of luminal/basal cells of dorsal prostate lobe, in con-
trast, it was absent in the lateral prostate lobe (Figs 3I–L and
4I–L). High EtOH consumption had significantly increased
the immunolabeling for RARc compared with the control
(Table 2). There was no significant difference for RARc in
the UChA groups (Table 2).
Cellular Proliferation and Apoptosis
Cell proliferation and apoptosis index showed no signifi-
cant differences among the groups. Additionally, it was
noted that cell proliferation tended to be increased in dorsal
prostate lobe of the UChB rats (Fig. 5).
The amount of ingested EtOH by the UChA and UChB
groups remained within the expected range for these varieties
(Quintanilla et al., 2006). Retinol consumption was not sig-
nificantly different among the groups, thus confirming that
our findings were not a consequence of the consumption.
Also, it was observed that EtOH consumption led to a
decrease in plasma retinol concentration proportionally to
Fig. 2. Quantification of endogenous all-trans-retinoic acid levels in
plasma and prostate. (A) UChB/UChBCo groups and (B) UChA/UChACo
groups. Values are expressed as mean ± SEM (n = 10/group). *p < 0.05
Fig. 3. Immunolocalization of retinoic acid receptors (RARa, RARb, and
RARc) in the dorsal lobe of prostate. RARa (A–D), RARb (E–H), and RARc
(I–L). Sections were counterstained with hematoxylin. Bars: 20 lm.
Table 2. Frequency of Retinoic Acid Receptors (RARs) in the UCh Rat Prostate
UChBUChBCo UChA UChACo
DP (%) LP (%)DP (%) LP (%)DP (%) LP (%)DP (%) LP (%)
81 ± 6
26 ± 3
25 ± 5
85 ± 5
15 ± 2
83 ± 4
6 ± 2**
5 ± 0.6**
83 ± 2
13 ± 2
75 ± 4
7 ± 0.6
16 ± 3
81 ± 4
7 ± 0.4
66 ± 5
10 ± 2
9 ± 2
63 ± 11
9 ± 1
DP, dorsal prostate; LP, lateral prostate; ND, not detected.
**p < 0.01.
UChB (DP) versus UChBCo (DP). Data are expressed asmean ± SEM (n = 5/group). Kruskal–Wallis complemented by Dunn’s test.
FONTANELLI ET AL.
the EtOH ingestion. The increased mobilization of retinyl
esters, the form of retinol storage, from the liver to the circu-
lation and later distribution to other tissues or even the
increased retinol metabolism, may result in depletion of
plasma retinol after EtOH exposure (Grummer and Erdman,
1983; Sato and Lieber, 1981).
It has been demonstrated that vitamin A (retinol plus
retinyl esters) concentration increases in some tissues after
chronic EtOH consumption (Leo et al., 1986; Mobarhan
et al., 1991). However, we did not find any change in
prostate retinol concentration during EtOH consumption.
Recently, it was proposed that exposure to EtOH is
linked to increase STRA6 and cellular retinol binding pro-
tein I (CRBPI) levels, further contributing to an elevation
in retinol levels (Kane et al., 2010). Likewise, increased
retinol concentration in BPH was suggested to occur
because of a high CRBP concentration (Pasquali et al.,
1996). Thus, it is therefore probably that EtOH does not
alter the protein levels related to the maintenance of reti-
nol homeostasis, thereby not changing the prostate retinol
increase in plasma and prostate ATRA concentration. It
seems true that plasma ATRA increases with chronic EtOH
exposure in response to the accentuated retinol metabolism
(Grummer and Erdman, 1983; Kane et al., 2010). Besides
that, the increased Raldh1 activity may also contribute to an
increase in ATRA levels when stimulated by chronic EtOH
(Kane et al., 2010). In some tissues, the initial increase in
ATRA concentration caused by the chronic presence of
EtOH induces the enzymes of cytochrome P450 (CYP)
involved in the ATRA catabolism, resulting in normal con-
centration of the acid with extended EtOH exposure (Kane
et al., 2010). We suggest that EtOH might be interfering in
ATRA synthesis or catabolism in the prostate through Ral-
dhs and CYPs. However, low EtOH consumption did not
changed in the plasma or prostate ATRA concentration.
Kane and colleagues (2010) have not found changes in
ATRA concentration after exposure to low amount of
EtOH, independent of exposure time or tissue analyzed.
Moreover, it is possible that the low EtOH consumption is
not sufficient to alter the enzymes involved in synthesis and
catabolism of ATRA in plasma and prostate.
The ATRA gene expression modulates and controls bio-
logical pathways such as cellular growth, epithelial differenti-
ation, and apoptosis through of RARs (Aboseif et al., 1997;
Fields et al., 2007). The RARs expression depends on the
amount of retinoids (retinol and retinoic acid) in prostate
(Pasquali et al., 1996, 1999). High EtOH consumption
increased ATRA levels in prostate as well as RARb and
RARc in dorsal prostate lobe, however, did not alter RARa
Fig. 4. Immunolocalization of retinoic acid receptors (RARa, RARb, and
RARc) in the lateral lobe of prostate. RARa (A–D), RARb (E–H) located in
the nucleus and cytoplasm of basal cells (arrow head) and RARc (I–L) dis-
playing no positive signal. Sections were counterstained with hematoxylin.
Bars: 20 lm.
Fig. 5. Cell proliferation and apoptosis in the prostate epithelial cells.
(A) Immunoreactions for cell proliferation (Ki-67) and (B) apoptotic cells
assay (TUNEL). Valuesare expressed as mean ± SEM. p > 0.05.
ETHANOL ALTERS ATRA/RARs ON THE PROSTATE
in dorsal and lateral prostate lobes. De The and colleagues
(1989) and Jones and colleagues (1997) found no differences
in the expression of RARa after treatment with retinoic acid.
The increase in ATRA concentration could differentially
stimulate its receptors, being RARa the most resistant to sen-
Steroid hormones also regulate RARs expression (Prins
et al., 2002). Huang and colleagues (1997) observed an
increase in RARa and RARc expression in prostate after
castration, while testosterone replacement had returned to
normal levels. EtOH exposure is known to decreases the con-
centration of testosterone in Wistar (Salonen and Huhtani-
emi, 1990), UChA, and UChB rats (Martinez et al., 2000).
Although EtOH consumption is linked to low testosterone
levels in both UChA and UChB rats, and this event seems to
increase RARs expression, our results pointed to an increase
in the RARb and RARc only in prostate of UChB. There-
fore, it suggest that increase of ATRA concentration in
UChB rat prostate results in overexpression of RARb and
RARc, being the dorsal prostate lobe more sensitive than
lateral prostate lobe of the prostate.
Changes in RARs may indicate deregulated cell growth or
even tumorigenic transformation of prostate epithelial cells
(Richter et al., 2002). The RARb is absent in prostate cancer
(Lotan et al., 2000), but increase in BPH (Richter et al.,
2002). The RARc also increase in BPH, but not alter in the
intraepithelial neoplasia and low-grade tumors. Thus, it
seems clear that alterations of RARs expression can be used
to differentiate benign from malignant tumors(Richter et al.,
Differences observed in RARb and RARc considering the
dorsal prostate lobe of UChB rats are similar to those
observed in BPH. Additionally, as occurred during the high
consumption of EtOH, the retinoic acid concentration also
increases in BPH (Pasquali et al., 1996). It is possible that
the high EtOH consumption and BPH acts through similar
pathways by modifying the prostatic homeostasis.
The maintenance of ATRA homeostasis is important
because cellular growth, epithelial differentiation and apop-
tosis rely on its concentration (De Luca, 1991). Retinoic acid
is also required for the development, differentiation
(Lasnitzki and Goodman, 1974; Vezina et al., 2008), and
maintaining homeostasis of epithelial cells in the adult pros-
tate (Prins and Putz, 2008). EtOH consumption decreases
the concentration of ATRA, resulting in increased cell prolif-
eration in the liver (Chung et al., 2001; Wang et al., 1998).
Our results showed that high EtOH consumption increases
the ATRA concentration in prostate, but not altering the cell
proliferation. It is probable that the increase in ATRA was
not sufficient to cause changes in cell proliferation.
Although the cell proliferation index has not presented sig-
nificant differences, high EtOH consumption led to a higher
cell proliferation index in the dorsal prostate lobe. EtOH has
been suggested as initiator for focal epithelial proliferation in
UChB rat prostate (Ca ˆ ndido et al., 2007). Moreover, EtOH
and acetaldehyde induce the transcription of activator prolif-
eration protein-1, in vitro, resulting in increased cell prolifer-
ation (Casini et al., 1994).
The proliferation and cell death index are normally
balanced in the adult prostate so that overgrowth does not
occur (Isaacs, 1984). However, the increased growth of the
prostate may result in BPH and prostate carcinoma. Several
reports have suggested that BPH can be chemically induced
in rodents (Ho et al., 1995; Scolnick et al., 1994). It should
be noted that high EtOH consumption promotes cell prolif-
eration more apparent in the dorsal lobe than lateral lobe of
prostate. Moreover, the increase in EtOH-related cell prolif-
eration could contribute to BPH. Nevertheless, further
studies are needed to clarify this hypothesis.
Similarly to Cagnon and colleagues (2001), EtOH con-
sumption did not alter the apoptotic index of prostate epithe-
lial cells. Additionally, there was no relationship between the
increased ATRA concentration and apoptosis. On the other
hand, retinoic acid is used as a chemotherapeutic agent to
induce apoptosis and differentiation in tumors (Cerniano
et al., 2008; Pahlman et al., 1984); however, these results are
achieved when using at high concentrations (Huss et al.,
2004; Leithner et al., 2000). We therefore suggest that the
increase in ATRA concentration arising from high EtOH
consumption is unable to interfere with the regulation of
over a longer period of EtOH exposure and therefore more
time of exposure to retinoic acid excess, because superphysio-
logical ATRA elevation contributes to EtOH-induced disor-
Additionally, changes in ATRA concentration and RARs
levels describe a critical event to prostate, because alterations
normal retinoic acid metabolism and RAR expression are
involved in both BPH and prostate cancer development
(Kim et al., 2005; Pasquali et al., 1996, 1999).
In summary, the present study demonstrated that EtOH
alters the plasma retinol concentrations proportionally to the
amount of EtOH consumed, whereas in the prostate, the reti-
nol concentration is not altered. Conversely, high consump-
tion of EtOH increases the concentration of ATRA in
plasma/prostate tissue and especially induces the RARb and
RARc in the dorsal prostate lobe. In addition, cell prolifera-
tion and apoptosis in the prostate does not change with
EtOH consumption and increased ATRA levels.
The study was supported by Coordenac ¸ a ˜ o de Aperfeic ¸ oa-
mento de Pessoal de Nı´vel Superior (CAPES).
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high-performance liquid chromatography. J Chromatogr 572:102–116.
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