original research:
First steps towards a new screening method for anabolic androgenic agents in human hair follicle
Martina Reiter1, Sybille Lüderwald2, Michael W. Pfaffl1, Heinrich H.D. Meyer1
1 Physiology Weihenstephan, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising, Germany,
2 Institute of Forensic Medicine, Universität München, Nussbaumstr. 26, 80336 München, Germany
Martina Reiter, Physiology
Weihenstephan, TU München, Weihenstephaner Berg 3, 85354 Freising,
martina.reiter@wzw.tum.de, Tel.:+498161713867, Fax:+498161714204
email: martina.reiter@wzw.tum.de
Submitted: 27 May 2008 | Accepted: 11 November 2008 | Published online: 15 Decembery, 2008
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Copyright © 2008 by Martina Reiter and colleagues, licensee The Doping Journal
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ABSTRACT
The
idea of this feasibility study in hair follicle was to test a possible
new way of detecting anabolic steroids, based on biomarker measurement
for different anabolic agents. With specific biomarker expression
patterns it should be possible to foster the existing doping analysis
methods. For the experiment the hair follicle was chosen as suitable
tissue.
The first aim was to develop a method for RNA isolation out of plucked
hair follicle samples. In a first study samples from untreated women
and men were taken to find gender specific differences and in a second
study also samples from weight lifters under doping conditions were
compared with a control group. As biomarkers, different androgen
dependent target genes were analyzed via qRT-PCR. Hair follicle cells
from the root sheath could be taken by plucking. In the first study no
significant gender dependent differences could be detected. In the
second study a down-regulation of the glucocorticoid receptor could be
calculated and fibroblast growth factor 7 was only expressed in treated
samples. These results show first promising differences between treated
and untreated samples.
INTRODUCTION
The
various doping scandals of the cyclist, especially during the “Tour de
France 2007” clearly showed that doping is a big topic in competitive
sport and doping controls need to be more universal in detecting drug
misuse. Beside doping methods that increase the oxygen transport
capacity in blood, especially the use of anabolic androgenic agents
(AAS) is very popular. These substances are supposed to increase muscle
mass via nitrogen retention resulting in a better performance [1, 2].
The International Olympic Committee (IOC) has already banned the use of
AAS by athletes in 1974 and methods for detecting and identifying these
substances are developed continuously. Most of them are
mass-spectrometry based, but there are still problems with the doping
control procedure. Tissues like urine, blood and hair are taken for
these analyses. They are easy to be collected and include the drugs or
its metabolites. Taking the existing analysis methods the question how
to screen for unknown designer drugs and its metabolites remains open.
It would be helpful to have additional methods which broaden the
existing screening spectrum [3, 4].
The effects of xenobiotic steroids in the organism are well documented
and it is known that these testosterone analoga can act via mRNA
expression in the cell, influencing the expression of different genes [5].
In the hair follicle androgenic hormones act via the dermal papilla but
also initiate different expression pathways in the cells of the inner
and outer root sheath [6].
The idea of this feasibility
study was to find a way to extract RNA from cells of hair follicle
samples obtained by plucking the hair of the androgen dependent frontal
scalp. The identification of different target genes could then be a
possibility to identify a biomarker pattern. Taking this androgen
specific pattern the intake of illegal drugs like testosterone and
other xenobiotic steroids could be possible.
To make sure that cells of the
hair follicle can be extracted by plucking the hair, histological
slices were gained and stained to identify the cell types of the hair
follicle.
Possible biomarkers (target genes) which are known to be influenced by
anabolic agents and which are expressed in the dermal papilla or hair
follicle were taken and separated in functional groups: Different
studies have mentioned the important role of estrogens in the hair
cycle, especially the estrogen receptor alpha (ERa) that mediates catagen induction of 17b-estradiol
in the hair follicle. It could be detected not only in the dermal
papilla cells but also in matrix keratinocytes and inner and outer root
sheath [7, 8]. Two other
important receptors of the AAS signaling pathways in the dermal
papillae are the androgen receptor (AR) and the glucocorticoid receptor
alpha (GRaaaaa). Through the binding to the receptors the androgens induce the mRNA expression of different genes [9, 10].
Factors that inhibit hair
follicle growth are the fibroblast growth factor 2 (FGF2) which is
expressed in the hair follicle and interleukin 1b (IL1b)
that is located in the epidermis and cells of the root sheath.
Fibroblast growth factor 7 (FGF7) is the antagonist and induces hair
follicle growth [11, 12].
Also apoptosis factors play a crucial role in the catagen phase of the
hair growth. Bcl-2 was found to be continuously expressed in the hair
papilla and plays a role as a support and signal center, and apoptosis
related receptors like FasR were analyzed in the follicle epithelium.
Also other factors of the extrinsic apoptosis pathway Caspase 3 and 8
were tested [6].
5a-reductase (SRD5A) is the enzyme that catalyses the transformation of testosterone to 5a-dihydrotestosterone,
an androgen that is supposed to be a mediator of hair loss. Two
isoenzymes type 1 and type 2 exist whereas type 1 is mainly expressed
in dermal papilla cells, type 2 also in dermal fibroblast. Additionally
5a-reductase type 2 is restricted to beard and frontal scalp [9, 13, 14].
This study was done to show up
significantly regulated target genes under the influence of anabolic
agents in hair follicles. Showing up the difference in the gene
expression regulations of samples from treated and untreated persons
should make it possible to identify the intake of specific anabolic
agents. Such specific biomarker expression patterns could foster the
existing analysis methods for anabolic agents in doping control and
help to uncover illegal abuses.
MATERIALS AND METHODS
HAIR FOLLICLE EXTRACTION
Frontal
scalp hair follicle samples were taken from the androgen dependent
upper part of the head. In the first study five hair follicle samples
were taken from women and five samples from men to find possible gender
specific differences. In the second study three samples from weight
lifters that are known to take different anabolic agents (testosterone,
trenbolone) could be taken and three hair samples from untreated man
served as control.
To get the hair follicle, five to six hairs were penned in a clamp,
plucked and cut to ca. 1 cm length. The hairs were immediately given in
a tube filled with lysis buffer (MasterPure RNA Purification Kit,
Epicentre Biotechnologies, Madison, WI, U.S.A.) and shock frozen in
liquid nitrogen. After freezing the samples were stored at -80°C.
To extract the RNA from the hair samples, the MasterPure RNA
Purification Kit and protocol (Epicentre Biotechnologies, Madison, WI,
U.S.A.) was used.
To quantify the amount of the extracted total RNA, optical density (OD)
was measured with the photometer (Eppendorf Biophotometer, Hamburg,
Germany). RNA purity was calculated with the OD260/280 ratio.
RNA QUALITY
To
get an idea about the RNA quality of the extracted total RNA, RNA
integrity and quality control was performed with the Bioanalyzer 2100
(Agilent Technology, Palo Alto, USA). Samples from both studies were
analyzed and taken as reference for an average RNA quality. For sample
analysis eukaryotic total RNA Nano Assay (Agilent Technology) was taken
and the RNA Integrity Number (RIN) served as RNA quality parameter [15].
PRIMER DESIGN AND TESTING
All primers were designed using published nucleic acid sequences of
Ensembl Genom Browser (http://www.ensembl.org) and NCBI
(http://www.ncbi.nlm.nih.gov). Primer 3 (http://frodo.wi.mit.edu/) was taken
to design and optimize all prime pairs with regard to primer dimer
formation, self-priming formation and primer annealing temperature at
60°C (Table 1). Designed primers were ordered and synthesized at MWG
Biotech (Ebersberg, Germany). All selected target genes were first
established in a cell culture study for HFDPC (hair follicle dermal
papilla cell) samples (Reiter et al., manuscript submitted).
TABLE 1
List
of target and reference genes measured in the hair follicle samples,
showing one specific product in the melt curve analysis. All other
genes showed unspecific primer dimers in the melt curve analysis.
Annealing temperature (AT) and the accession number in NCBI (Acces.Nr.)
are listed
Note: you may need
to resize your browser window for better view of Table 1
REAL-TIME qRT-PCR
Quantitative real-time RT-PCR was performed in the Rotor Gene 6000
(Corbett Life Science, Sydney, Australia) using SuperScript III
Platinum SYBR Green One-Step qPCR Kit (Invitrogen, Carlsbad, USA) by a
standard protocol. Samples were diluted to 10ng/µl and 50ng tRNA in 5µl
(Invitrogen, Carlsbad, USA) was added to each sample to protect mRNA of
degradation. 38 ng total RNA were taken for one PCR reaction in a total
volume of 10µl. The PCR protocol had following steps: Hold 1 (55°C,
10min), Hold 2 (95°C, 5min), Cycling (40 cycles: 95°C, 15sec; 60°C,
20sec; 68°C, 20sec), Melt curve (60°C- 99°C, 0.5°C/step). Crossing
points (Ct) and melting curves were acquired by using the
“quantitation” and “melting curve” program of the Rotor-Gene 6000
analysis software.
Only genes with clear and single melting peaks were taken for further
data analysis. Samples with irregular melting peaks were excluded from
the calculation. All samples were baseline corrected and threshold was
set manually, using same threshold levels for one gene in all samples.
HISTOLOGICAL STAIN AND IMMUNOHISTOCHEMISTRY
Hair samples were incorporated in frozen section medium (Richard-Allan
Scientific, Kalamazoo, USA) and prepared using a cryostat mycrotom HM
505E (Micron, Walldorf, Germany). 6 µm slices of the hair follicles
were fixed on the object holder with 100%, -30°C frozen ethanol and
stained in haematoxylin (Sigma, Germany) for 3 min. The histological
slices were blued with tap water, stained in 1% eosin (Sigma-Aldrich,
Munich, Germany) for 7 min, washed with 50-100% ethanol and finally
given in rotihistol (Sigma-Aldrich, Munich, Germany). With EUKITT the
cover slips were fixed on the object holder and hardened over night.
DATA ANALYSIS AND STATISTICS
Data were processed applying relative quantification method comparable
to the ddCt-method (2ddCt) [16]. For normalization of target gene (TG)
expression the arithmetic mean of the following non regulated reference
genes (RG) were taken for the calculation of a reference gene index
(RGI): In study one (RG1) glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) and (RG2) ubiquitin (UBQ), in study two (RG1) ACTB and (RG2)
UBQ. For every sample gene expression of the two RG were analyzed and
the mean value served as reference gene index.
In Excel (Microsoft, USA) all calculations were done. T-test
calculations were used to find gender specific regulations of the
target genes. In study one only Ct could be calculated because no
control group existed. In study two ddCt and the expression ratio could
be analyzed.
RESULTS
HISTOLOGICAL STAIN
Hair samples were incorporated in frozen section medium (Richard-Allan
Scientific,
Twenty
males participated in this study. Subjects mean (SD) age, resting SBP,
DBP and HR and were 22.3 (4.0) years, 127 (5.4) mmHg, 78 (4.2) mmHg and
70 (4.8) beats/minute, respectively. The mean run time, no of exercise
laps and VO2 max are 8 (2.0) mins, 72.8 (8.0) m and 42.5 (5.0)
ml.kg-1.min-1 respectively. Detailed physical characteristics are
depicted in Table 1.
xfig1
FIGURE 1
Slice
of the plucked hair with the hair shaft (1) and outer root sheath cells
(2) which are clearly outlined by perifollicular sheath cells (3),
stained in blue.
Note: you may need
to resize your browser window for better view of Figure 1
RNA CONCENTRATION AND QUALITY OF HAIR FOLLICLE SAMPLES
Mean
total RNA concentration of all samples of the first study was
674.6±520 ng, 741.4±546.6 ng in male and
676.8±509.7 ng in female samples. In the second study mean RNA
concentration was 1144±64.4 ng.
The measured RIN values showed and average value of 7.9 what is high enough to expect good results in qRT-PCR.
FIGURE 2
Melt
curve of FGF7 in study 2 with the melt temperature on the x-axis and
the first derivative of the melt curve on the y-axis. Control samples
(green) do not show a specific product, treated samples (orange) show a
specific melt peak at 83°C
Note: you may need
to resize your browser window for better view of Figure 2
PCR CONCENTRATION OF HAIR FOLLICLE SAMPLES
In
the first study the reference genes GAPDH and UBQ and in the second
study the reference genes ACTB and UBQ were not significantly regulated
and could be taken for statistical calculation. In the first study the
target genes AR, ERa, FGF2, FGF7, IL1, SRD5A2, FasR, Bcl-2, Caspase 8 and 9 were measured, in the second study AR, GRa, ERa, FGF7, FasR, Caspase 3 and 8. The melt curve analysis of AR, ERa,
SRD5A2 and Caspase 8 did not show a specific product. FGF7 only showed
a specific product in the treated samples of study two, in the control
samples the melt curve analysis showed no specific peak (Figure2). The
melt curve of GR showed a specific peak in the treated and untreated
samples, with better results in peak height showing the sensitivity of
the amplification in the treated samples (Figure 3).
FIGURE 3
Melt
curve of GR in study 2 with the melt temperature on the x-axis and the
first derivative of the melt curve on the y-axis. Control samples
(purple) show slightly lower peak heights at 86°C than treated
samples (green)
Note: you may need
to resize your browser window for better view of Figures
DATA ANALYSIS AND STATISTICS
In
study 1 none of the measured target genes showed significant
differences (p-value<0.05) between the male and female samples in
the first study. In the second study a significant down-regulation of
41 % for the GRa could be calculated (p-value=0.02). For FGF7 the
treated samples had a mean Ct of 23.9 cycles and a mean dCt-value of 4.88. This shows a lower expression compared to the reference genes.
DISCUSSION
Different
studies have shown that anabolic agents influence hair growth through
the dermal papilla via regulation of different genes in males and
females. The gene expression regulations induced in the papilla also
showed effects on growth factors of root sheath cells [17, 18, 19].
Analyzing these specific regulations in hair follicle samples could be
a new possibility to identify the intake of forbidden anabolic agents
like testosterone and other xenobiotic.
Histological stains of plucked hair follicle samples clearly showed
that cells of the root sheath, especially the outer root sheath could
be gained and used for RNA extraction without taking a skin biopsy.
This is a pre-condition for the use in doping analysis because sampling
has to be simple practicable. Samples from the androgen dependent part
of the scalp (frontal scalp hair) were taken to show possible gender
specific differences in gene expression. It was not possible to gain
the hair papilla by plucking as expected. This could explain that AR, ERa and SRD5A2, target genes that are known to be mainly expressed in the hair papilla could not be detected.
To get hair follicle samples from weight lifters that own up to take
anabolic agents is quite difficult; therefore only three samples could
be analyzed and taken for statistical analysis. However, first
significant differences between the treated and untreated samples could
be detected. A first androgen dependent effect could be seen in the
expression of FGF7 that could only be measured in the samples of the
treated weight lifters, all untreated samples from study one and two
did not show a specific product in qRT-PCR analysis.
It is known that androgens can bind and act via the GR what can be
confirmed by the significant down-regulation of the receptor and the
higher amplification sensitivity in the treated samples, in the second
study.
The expression of IL1b and
Bcl-2 in the first study and FasR in both studies in the inner and
outer root sheath of the hair follicle could be approved [6, 10, 20].
CONCLUSION
With
this study a first step towards a possible new screening method for
anabolic androgenic agents via gene expression analysis could be done,
with the aim to support existing, mass spectrometry based detection
methods. Plucking hairs and taking the RNA from the hair follicle seems
to be a practicable method to analyze different gene expression and
find specific biomarkers. A sampling and extraction method could be
successfully developed and first target genes could be analyzed.
Differences in gene expression regulations between the different
genders could not be calculated. It seems that only very high androgen
concentrations significantly influence these gene regulations. This
effect could be seen in the expression of FGF7 and the significant
regulation of GR only in samples of androgen treated athletes, what
makes these two target genes to first biomarkers of AAS.
These results show first promising differences between treated and
untreated samples, a precondition for the development of a possible
screening method for anabolic agents. The ambitions for the future
should be the identification of specific biomarker patterns for
functional groups of anabolic agents, in order that abuses can be
uncovered.
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article should be cited in the following way:
M. Reiter, S. Lüderwald,
M.W. Pfaffl, H.H.D. Meyer. First steps towards a new screening method
for anabolic androgenic agents in human hair follicle?
The Doping Journal
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ABSTRACT