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Research Article
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Berberine Disturbs the Expression of Sex-hormone Regulated Genes in β-naphthoflavone-induced Mice
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Thinnakorn Lao-Ong,
Waranya Chatuphonprasert
and
Kanokwan Jarukamjorn
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ABSTRACT
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Berberine is a major active constituent in several medicinal plants such as Coscinium fenestratum, Berberis aristata, Coptis japonica and Coptis chinensis. Berberine possesses a wide range of biochemical and pharmacological activities including anti-inflammation, anti-microorganism, anti-cancer activities and anti-diabetes. In this study, we determined the effect of berberine, in combination with β-naphthoflavone (BNF, a pro-carcinogen), on the expression of sex-hormone synthesis genes at mRNA level including CYP17, CYP19, 3β-HSD, 17β-HSD1, 17β-HSD3 in mouse testes. The single treatment of berberine up-regulated the expression of testicular CYP17, 3β-HSD and 17β-HSD1 mRNA. BNF up-regulated only 17β-HSD1 mRNA. Moreover, the combination of berberine and BNF up-regulated the expression of CYP17 and 3β-HSD. These observations suggested that berberine might disturb sex-hormone synthesis pathway; consequently possibly resulted in modification of estrogen or testosterone synthesis. Therefore, a caution should be noted for the use of berberine as an alternative medicine, especially at high dose or long period.
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Received:
March 08, 2013; Accepted: March 25, 2013;
Published: July 10, 2013 |
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INTRODUCTION
In steroidogenesis (Fig. 1), the first step is converting
cholesterol into pregnenolone by cytochrome P450 (CYP11α) encodes cytochrome
P450scc (cholesterol side-chain cleavage enzyme) (Miller,
1988; Stocco, 2000) Afterwards, pregnenolone is
converted to progesterone by 3β-hydroxysteroid dehydrogenase (3β-HSD).
The human cytochrome P450c17α enzyme (CYP17) possesses two enzymatic actions
namely 17α-hydroxylase and 17,20-lyase which participate in two different
catalytic steps in the steroid hormone synthesis pathway (Zuber
et al., 1986). Pregnenolone and progesterone are converted by 17α-hydroxylase
to 17-hydroxypregnenolone and 17-hydroxyprogesterone, respectively. After that,
the 17,20-lyase turns both 17-hydroxypregnenolone and 17-hydroxyprogesterone
into dehydroepiandrosterone (DHEA) and androstenedione, respectively. The expression
of human CYP17 is constitutively expressed in female ovarian theca cells and
adrenal cortex. Rare mutations in the coding region of CYP17 have been found
in patients with 17α-hydroxylase/17,20-lyase deficiency, resulting in various
clinical profiles such as congenital adrenal hyperplasia, abnormal sexual development,
osteoporosis and irregular menstruation (Yanase et al.,
1991; Yanase, 1995). The 3β-hydroxysteroid
dehydrogenase-isomerase enzyme (3β-HSD) functions by converting pregnenolone
to progesterone. Moreover, 3β-HSD can convert progesterone and DHEA to
androstenedione (Bates et al., 2005). The aromatase
P450 enzyme (CYP19) is a catalyzer for the conversion of androstenedione to
estrone, or modification of testosterone to estrogen in many tissues (Morishima
et al., 1995). The reproductive systems of rats, mice and human are
affected by aromatase (CYP19) inhibition. Deficiency of CYP19 enzyme in male
mice severely disturbed spermatogenesis resulted in infertility and consequently
deteriorated ability to breed pups. In females, CYP19 deficiency likewise resulted
in lack of estrogen followed by pseudohermaphroditism and masculinization in
adolescence (Carani et al., 1997).
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Fig. 1: |
Sex-hormone synthesis pathway |
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Fig. 2: |
Structure of (a) Berberine and (b) β-naphthoflavone |
The latter pathway culminates in the formation of testosterone via conversion
of androstenedione by 17β-hydroxysteroid via conversion of androstenedione
by 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) while 17β-hydroxysteroid
dehydrogenase type 1 (17β-HSD1) converts the formation of estrone to estradiol
(Sha et al., 1996).
Berberine (Fig. 2) is a major plant alkaloid present in several
Indian and Chinese herbal plants; Coscinium fenestratum, Berberis
aristata, Berberis vulgaris, Hydrastis Canadensis, Tinospora
cordifolia, Coptis japonica and Coptis chinensis (Rojsanga
et al., 2006; Sato and Yamada, 1984). The
extensive researches demonstrated a number of biological and pharmacological
benefits of berberine, i.e., anti-inflammation (Kuo
et al., 2004), anti-microorganism (Schmeller
et al., 1997), anti-cancer activities (Wu et
al., 1999; Letasiova et al., 2006; Piyanuch
et al., 2007) and anti-diabetes (Zhang et
al., 2008; Zhang et al., 2011).
Our previous study noted the effects of berberine on β-naphthoflavone
(BNF)-induced CYP1A expression in both primary mouse hepatocytes and mouse livers
(Chatuphonprasert et al., 2011). The BNF-induced
expressions of mouse CYP1A mRNA and protein and the related enzyme activities
were significantly suppressed by berberine. In addition, berberine significantly
lowered the level of BNF-induced lipid peroxidation in mouse hepatic microsome
(Chatuphonprasert et al., 2011). The study suggested
that the use of berberine as an alternative medicine might bring more advantages
due to its ability to decrease the risk of carcinogenesis from induction of
CYP1A expression and inhibitory effect on lipid peroxidation activity. In this
study, we determined the effect of berberine, in combination with BNF (as pro-carcinogen),
on the expression of sex-hormone synthesis genes at mRNA level including CYP17,
CYP19, 3β-HSD, 17β-HSD1, 17β-HSD3 in mouse testes. Completeness
of the study provided more detail of utilization of berberine. In contrast,
if there is any negative effect found, precaution of berberine would be concerned.
MATERIALS AND METHODS
Chemicals: Berberine choride (Ber), β-Naphthoflavone (BNF) and
corn oil were supplied by Sigma-Aldrich Chemical (St. Louis, MO). ReverTraAce
was a product of Toyobo (Osaka, Japan). Trizol® reagent, random
primers (hexadeoxyribonuclotide mixture), Taq DNA polymerase, RNase inhibitor
and dNTP mixtures were products of InvitrogenTM (Carlsbad, CA). All
other laboratory chemicals were of the highest purity available from commercial
suppliers. Forward and reverse primers of mouse CYP17, CYP19,
3β-HSD, 17β-HSD1, 17β-HSD3 and GAPDH genes
were synthesized by Bio Basic, Inc. (Markham Ontario, Canada). The primers of
each gene are shown in Table 1.
Table 1: |
Primers sequences for PCR |
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Table 2: |
Conditions of PCR cycles |
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Animals and treatments: Male C57BL/6 mice were supplied by National
Laboratory Animal Center, Mahidol University, Nakhon Pathom, Thailand. All mice
were housed in the Animal Unit of Faculty of Pharmaceutical Sciences, Khon Kaen
University. Mice were housed in the Northeast Laboratory Animal Center (Khon
Kaen University, Khon Kaen, Thailand) under the supervision of a certified laboratory
veterinarian. They were treated according to a research protocol approved by
the Animal Ethics Committee for Use and Care of Khon Kaen University, Khon Kaen,
Thailand (AEKKU 06/2553). At all times, the mice were housed on wood chip bedding
in polysulfone cages with water and commercial animal diet supplied ad libitum
and were acclimated for at least 7 days in housing with a 12 h dark/light cycle
under controlled temperature (23±2°C) and humidity (45±2%)
before dosing. Seven week-old mice were orally fed daily with 7.5 mg/kg/day
of berberine (Ber) for 7 days and/or intraperitoneally given 30 mg/kg/day of
BNF in the last 3 days. The control group was orally given corn oil daily for
7 days. The mice were sacrificed at the 24 h after the last treatment and the
livers were excised immediately for preparation of total RNA from testes.
Semi-quantitative reverse transcription polymerase chain reaction: Mouse
CYP17, CYP19, 3β-HSD, 17β-HSD3, 17β-HSD1 and GAPDH mRNAs were
semi-quantified by RT-PCR. Testicular total RNA was reverse-transcribed using
random primer and ReverTraAce®, then cDNA was amplified. The
conditions of PCR cycle were followed by the method of Degawa
et al. (2006), Sha et al. (1996),
Udomsuk et al. (2011) and Chatuphonprasert
et al. (2009) with some modifications (Table 2).
After separation of the PCR products by 2% agarose gel electrophoresis, the
target cDNA bands were detected under ultraviolet light in the presence of ethidium
bromide and semi-quantified by Gel documentation (Ingenius syngene bio-imagimg,
model: IngeniusL) and Gene Tool Match program (Syngene, Lab Focus Co. Ltd.,
Cambridge, UK). The mRNA levels of the target genes were normalized to that
of a house keeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
RESULTS AND DISCUSSION
Modification of sex-hormone related genes including CYP17, 3β-HSD,
CYP19, 17β-HSD3 and 17β-HSD1 and GAPDH,
at the transcriptional level after treatment with berberine and/or BNF were
examined in testes. The single treatment of berberine and the co-treatment with
BNF significantly up-regulated expression of CYP17 and 3β-HSD mRNA (Fig.
3). Polymorphism of CYP17 gene influences breast cancer in young
women and up-regulated transcription of CYP17 might affect the synthesis of
estrogen and testosterone (Bergman-jungestrom et al.,
1999). In addition, the retinoids stimulated mRNA abundance and promoter
function of CYP17 was found in polycystic ovary syndrome theca cell (Wickenheisser
et al., 2005). Hence, the enhancing effect of berberine and the co-treatment
with BNF might be aware due to CYP17 induction effect. 3β-HSD involves
in several step of sex-hormone synthesis pathway (Zuber
et al., 1986; Bates et al., 2005).
Therefore, berberine might disturb sex-hormone synthesis, consequently possibly
resulted in modification of circulating pattern of estrogen or testosterone
in the body. Moreover, the expression of 17β-HSD1 mRNA was significantly
up-regulated after single treatment with berberine or BNF but there was no change
in co-treatment group (Fig. 4). The up-regulated expression
of 17β-HSD1 might be involved in estradiol metabolism pathway by an increase
the conversion of estradiol.
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Fig. 3: |
Expression pattern of testicular CYP17 and 3β-HSD mRNA
in male mice Seven week-old mice were orally fed daily with 7.5 mg/kg/day
day of berberine (Ber) for 7 days and/or intraperitoneally given 30 mg/kg/day
day of β-naphthoflavone (BNF) in the last 3 days. The control group
(NT) was orally given corn oil daily for 7 days. The relative mRNA expression
levels were normalized by that of GAPDH. The data are presented as the Mean±SD
(n = 3-4) from at least 2 independent experiments. A significant difference
was examined by ANOVA with Tukey post hoc test. *p<0.05, **p<0.001
compared to NT group; #p<0.05 compared to BNF group |
Hepatic cytochrome P450 1A1 (CYP1A1) and cytochrome P450 1B1 (CYP1B1) play
key roles in catalyze estrogen to 2 and 4-hydroxyestradiol and convert to methoxyestradiol
by catechol-O-methyltransferase (Liehr, 2000; Dawling
et al., 2003). The previous study showed that BNF induced CYP1A1
and CYP1B1 activities. Activations of these enzymes lead to increase estradiol
metabolism and resulted in up-regulated expression of 17β-HSD1. The unchange
of 17β-HSD1 by the co-treatment resulted from the feedback inhibition of
estradiol metabolism by methoxyestradiol (Liehr, 2000;
Dawling et al., 2003). The results in estradiol
accumulation accordance with 17β-HSD1 recovered to nearly the same level
as the normals (Fig. 4). The expressions of CYP19 and 17β-HSD3
were not significantly modified by all treatments (Fig. 5).
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Fig. 4: |
Expression pattern of testicular 17β-HSD1 mRNA in male
mice, Seven week-old mice were orally fed daily with 7.5 mg/kg/day of berberine
(Ber) for 7 days and/or intraperitoneally given 30 mg/kg/day of β-naphthoflavone
(BNF) in the last 3 days. The control group (NT) was orally given corn oil
daily for 7 days. The relative mRNA expression levels were normalized by
that of GAPDH. The data are presented as the Mean±SD (n = 3-4) from
at least 2 independent experiments. A significant difference was examined
by ANOVA with Tukey post hoc test. * p<0.05, ** p<0.001 compared
to NT group; # p<0.05 compared to BNF group |
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Fig. 5: |
Expression pattern of testicular CYP19 and 17β-HSD3 mRNA
in male mice, Seven week-old mice were orally fed daily with 7.5 mg/kg/day
day of berberine (Ber) for 7 days and/or intraperitoneally given 30 mg/kg/day
day of β-naphthoflavone (BNF) in the last 3 days. The control group
(NT) was orally given corn oil daily for 7 days. The relative mRNA expression
levels were normalized by that of GAPDH. The data are presented as the Mean±SD
(n = 3-4) from at least 2 independent experiments. A significant difference
was examined by ANOVA with Tukey post hoc test. *p<0.05, **p<0.001
compared to NT group;#p<0.05 compared to BNF group |
CONCLUSION
In the conclusion, the single treatment of berberine up-regulated the expression
of testicular CYP17, 3β-HSD and 17β-HSD1 mRNA. The single treatment
of BNF up-regulated only 17β-HSD1 mRNA expression. While the combination
of berberine and BNF up-regulated in expression of CYP17 and 3β-HSD. These
observations suggested that berberine disturbed sex hormone synthesis pathway;
consequently possibly resulted in modification of circulating estrogen pattern
in the body. Therefore, a caution should be noted for the use of berberine as
an alternative medicine. However, a further study to affirm the evidence of
sex hormone modification by berberine in illness is still required.
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