INORGANIC COMPOUNDS
Assessment of DNA integrity (COMET assay) in sperm cellsof boron-exposed workers
Yalcın Duydu • Nursen Basaran • Aylin Ustundag • Sevtap Aydın •
Ulku Undeger • Osman Yavuz Ataman • Kaan Aydos • Yalcın Duker •
Katja Ickstadt • Britta Schulze Waltrup • Klaus Golka • Hermann M. Bolt
Received: 5 July 2011 / Accepted: 25 July 2011 / Published online: 11 August 2011
� Springer-Verlag 2011
Abstract An extension of a male reproductive study
conducted in a boric acid/borate production zone at
Bandırma, Turkey, is presented. The relation between
DNA-strand breaks (COMET assay, neutral and alkaline
version) in sperm cells and previously described sperm
quality parameters was investigated in boron-exposed
males. A correlation between blood boron levels and mean
DNA-strand breaks in sperm was weak, and DNA-strand
breaks in sperm were statistically not different between
control and exposed groups. Therefore, increasing boron
exposures had no additional contribution in addition to
already pre-existing DNA-strand breaks in the sperm cells.
Weak but statistically significant correlations between
DNA-strand breaks and motility/morphology parameters of
sperm samples were observed in the neutral version of the
COMET assay, while correlations between the same vari-
ables were statistically not significant in the alkaline ver-
sion. A likely reason for these negative results, even in
highly exposed humans, is that experimental exposures that
had led to reproductive toxicity in animals were signifi-
cantly higher than any boron exposures, which may be
reached under realistic human conditions.
Keywords Boric acid � Borax � Boron � COMET assay �Fertility � Sperm parameters � Male reproduction
Introduction
Inorganic borates and boric acid display only low acute
toxicity, irrespective of the mode of administration. Upon
experimental repeated administration, there is similarity in
toxic effects across species of boric acid and of borates that
dissociate to boric acid. This provides a rationale to cal-
culate doses on an equivalent boron (B) basis (Hubbard
1998, WHO (World Health Organization) 1998). Repro-
ductive effects were recognized as the critical toxicity of B.
Hence, there is regulatory concern with regard to possible
human environmental and occupational exposures to boric
acid and borates (Murray 1995, Jensen 2009, Bonde 2010,
Soucek et al. 2011). Both boric acid and (sodium) borates
have been considered as toxic to reproduction (ECHA
(European Chemicals Agency) 2008). NOAEL values
underlying to current regulatory assessments are 9.6 mg
Electronic supplementary material The online version of thisarticle (doi:10.1007/s00204-011-0743-9) contains supplementarymaterial, which is available to authorized users.
Y. Duydu (&) � A. Ustundag
Department of Toxicology, Faculty of Pharmacy,
Ankara University, Tandogan, 06100 Ankara, Turkey
e-mail: [email protected]
N. Basaran � S. Aydın � U. Undeger
Department of Toxicology, Faculty of Pharmacy,
Hacettepe University, Sıhhiye, 06100 Ankara, Turkey
O. Y. Ataman
Department of Chemistry, Middle East Technical University,
06531 Ankara, Turkey
K. Aydos
Department of Urology, Faculty of Medicine, Ankara University,
06100 Ankara, Turkey
Y. Duker
Bandırma State Hospital, Bandırma, Turkey
K. Ickstadt � B. S. Waltrup
Faculty of Statistics, TU Dortmund, 44221 Dortmund, Germany
K. Golka � H. M. Bolt
Leibniz Research Centre for Working Environment and Human
Factors (IfADo), Ardeystr. 67, 44139 Dortmund, Germany
123
Arch Toxicol (2012) 86:27–35
DOI 10.1007/s00204-011-0743-9
B/kg per day and 17.5 mg B/kg per day in rats, for
developmental and male fertility effects, respectively
(Weir and Fisher 1972, Price et al. 1996, WHO (World
Health Organization) 1998). With respect to male fertility,
depressed testicular weight and testes atrophy linked with
an effect on spermatogenesis were addressed as most
sensitive experimental endpoints in rats (Ku et al. 1993).
The mode of action of the reproductive toxicity of B is still
a matter of speculation (Huel et al. 2004).
Already in 1998, it has been argued that B doses that
experimentally cause reproductive effects must be far
higher than any levels to which the human population
could be exposed, as humans needed to ingest some 3.3 g
of boric acid (or 5.0 g of borax) daily to arrive at dose
levels equivalent to the lowest animal NOAEL. It was also
argued that so far no effects on fertility had been seen in
humans occupationally or environmentally exposed to
borates (Hubbard 1998).
Very recently, this view was substantiated by two
independent studies in highly B-exposed workers. The first
study was performed in Chinese workers, in which semen
parameters (total sperm count, sperm concentration,
motility, morphology, DNA breakage, apoptosis and
aneuploidy) were not affected with increasing B exposure
(Robbins et al. 2010). Our group conducted a second study
in highly B-exposed workers in Turkey, and again no dif-
ferences in sperm concentration and sperm motility
parameters were noted between control and exposed groups
(Duydu et al. 2011). A detailed assessment of the effective
B doses and associated blood levels involved in the rele-
vant human and experimental studies is presented else-
where (Bolt et al. 2011).
In view of the current regulatory discussion on possible
male fertility effects due to B, our investigation in highly
B-exposed workers in Turkey was supplemented by an
assessment of DNA integrity in cryopreserved sperm cells
using the COMET assay protocol. This methodology is
backed by recent clinical experience in natural and assisted
fertilization. Clinical studies have made the point that
damage of sperm DNA, in combination with classical
sperm quality parameters, is predictive of reduced male
fertility (Lopes et al. 1998, Sakkas et al. 2000, Morris et al.
2002). So far, the only epidemiological study in a boron-
exposed cohort assessing both sperm DNA integrity (using
the COMET assay) and classical sperm quality parameters
was the study in Chinese B-exposed workers by Robbins
et al. (2010). Therefore, there is an obvious need of con-
firmation by a second independent study.
The present report describes an extension of our male
reproductive study conducted in a boric acid/borate pro-
duction zone at Bandırma, Turkey. Details concerning
exposure, sampling, and assessment of biomarkers and of
male reproductive parameters have been described
previously (Duydu et al. 2011). The application of the
COMET assay to sperm specimens collected at remote
sites and then transported to a central laboratory is by no
means trivial, as underlined by others (Young et al. 2003).
Therefore, special care was taken to ensure appropriate
quality control.
Materials and methods
The present study was approved by the Ethics Committee
of the School of Medicine, Hacettepe University (HEK
08/167, 22.10.2008). All study subjects gave informed
consent prior to participation in the project. Subjects were
male boron-exposed workers employed in boric acid pro-
duction plant, Bandırma, Turkey. The present report rep-
resents an extension of the main study published by Duydu
et al. (2011), to which reference is made concerning study
details and the local situation. The following briefly sum-
marizes the essentials.
Study cohort
All married workers wishing to participate were enrolled.
They agreed to provide the biological samples and to
complete a questionnaire with demographic, exposure,
reproductive and general health information. The total
number of workers working in production of boron prod-
ucts and therefore potentially exposed to B was 428. One
hundred and two of them participated in the study. The
total number of workers (male) working in the same zone,
but outside of the production area of boron products and
not occupationally exposed to boron, was 432. One hun-
dred and two of these participated in the study as workers,
who were not exposed occupationally. Subsequently, the
biological monitoring results showed that there was sig-
nificant environmental B exposure, due to pollution of the
drinking water supply of the industrial complex. Therefore,
the entire cohort (n = 204, including those not occupa-
tionally exposed) was re-classified according to the indi-
vidual B blood levels (for details, see Duydu et al. 2011):
(i) re-defined ‘‘control group’’ (n = 49), below the limit of
quantitation (48.5 ng B/g blood); (ii) re-defined ‘‘low
exposure group’’ (n = 72), above the limit of quantitation,
up to 100 ng B/g blood; (iii) re-defined ‘‘medium exposure
group’’ (n = 44), over 100 ng B/g blood, up to 150 ng B/g
blood; (iv) re-defined ‘‘high exposure group’’ (n = 39),
above 150 ng B/g blood.
Biological samples
Semen, blood and urine samples were obtained from the
voluntary employees. The biological monitoring methods
28 Arch Toxicol (2012) 86:27–35
123
for boron in biological fluids, as well as clinical chemistry
methods, were described previously (Duydu et al. 2011).
Sampling and analysis of the sperm samples
Semen samples were obtained from a total of 198 out of
204 boron-exposed men into sterile wide-mouthed con-
tainers at the infirmary of the boric acid production plant.
The workers were informed about the importance of
abstinence for at least 48 h (but no longer than 7 days)
prior to providing the semen samples (WHO (World Health
Organization) 2003).
After liquefaction at 37�C for 30 min, the semen sam-
ples were prepared for both morphologic and motility
analysis. Sperm concentration and motility parameters
were determined by using SQA-V Gold Sperm Quality
Analyzer within 1 h after sampling. In addition, duplicate
slides were appropriately prepared and stained for each
semen sample to analyze sperm morphology as per the
Kruger’s strict criteria (Kruger et al. 1986, 1988). These
analyses were performed by one experienced technician at
the Research Center on Fertility, School of Medicine,
University of Ankara. Methodological details and the
corresponding results were described previously (Duydu
et al. 2011).
Cryopreservation of the semen samples
After liquefaction at 37�C for 30 min, the semen samples
were aspirated into 0.3 mL cryostraws and sealed with
labeling rods provided by the manufacturer (Cryo-Bio-
System, France). The cryostraws immersed directly into
the liquid nitrogen (flash-freezing) and stored throughout
sampling and transportation period.
Thawing the sperm samples
After completing the sampling process in Bandırma, all
semen samples were transported to the laboratory of
Ankara University, Faculty of Pharmacy, Department of
Toxicology, in liquid nitrogen tanks. The straws were
thawed in phosphate-buffered saline (PBS) solution at
room temperature and then immediately processed for the
COMET assay in the same laboratory.
COMET (single-cell gel electrophoresis) assay
The DNA integrity of sperm cells was determined by using
alkaline and neutral single-cell gel electrophoresis assay
(Donnelly et al. 2001, Young et al. 2003). According to this
assay protocol, the cells were embedded in agarose gel,
lysed and fragmented DNA strands drawn out by electro-
phoresis to form a COMET.
Embedding of sperm in agarose gel
Slides were covered with 1% normal-melting point agarose
in Ca2?- and Mg2?-free phosphate-buffered saline. The
slides were left to allow the agarose to solidify. The sperm
suspension (1 9 105 cells/50 lL) was mixed with the
100 lL of 0.5% low-melting point agarose at 37�C. This
cell suspension was rapidly pipetted on top of the first
agarose layer, covered with a coverslip and allowed to
solidify at room temperature.
Lysing of cells
After solidification of the agarose gel, the coverslips were
removed and the slides were immersed in a coplin jar
containing freshly prepared cold lysing solution (2.5 mol/L
NaCl, 100 mmol/L Na2EDTA, 10 mmol/L Tris, pH 10,
with 1% Triton X-100 added just before use) for 1 h at 4�C.
Slides were then incubated for 30 min at 4�C with
10 mmol/L dithiothreitol followed by 90-min incubation at
20�C with 4 mmol/L lithium diiodosalicilate (Robbins
et al. 1993, Donnelly et al. 2001).
Unwinding of DNA and electrophoresis
Alkaline conditions The slides were removed from the lysis
solution and placed in a horizontal gel electrophoresis tank
filled with freshly prepared alkaline electrophoresis solu-
tion (300 mmol/L NaOH, 1 mmol/L Na2EDTA, pH 13.0).
The slides were left in this pH buffer for 20 min to allow
the DNA to unwind. Electrophoresis was then conducted
for 20 min by applying an electric current of 25 V/300 mA
to allow damaged DNA to migrate from the nucleus toward
the anode. The slides were than drained, placed on a tray
and washed with three changes of neutralization buffer
(0.4 mol/L Tris, pH 7.5) for 5 min each. The slides were
left to drain before staining.
Neutral conditions The slides were removed from the
lysis solution and placed in a horizontal gel electrophoresis
tank filled with freshly prepared neutral electrophoresis
solution (300 mmol/L sodium acetate, 1 mmol/L Tris, pH
9.0). The slides were left in this pH buffer for 20 min.
Electrophoresis was then conducted for 1 h by applying an
electric current of 18 V/100 mA. The slides were rinsed
gently with deionized water.
Staining and image analysis
The slides were stained with 50 lL (20 lg/mL) ethidium
bromide and covered with a coverslip. The slides were
viewed using a fluorescence microscope (Leica DM1000)
equipped with an excitation filter of 515–560 nm. A single
scorer randomly selected and captured 100 cells using the
Arch Toxicol (2012) 86:27–35 29
123
Perceptive Instruments COMET Assay IV analysis system.
Tail % intensity was selected as the image analysis
parameter.
Technical controls and quality assurance measures
Semen samples collected from 2 donors living in Ankara
(40, 45 years old, healthy, fertile, nonsmokers) were used
as quality assurance to check the intra-assay variability.
The total number of sperm in the freshly produced semen
samples was counted using the improved Neubauer
hemocytometer and aspirated into 10 (3 straws from one
donor and 7 straws from the other) cryostraws (0.3 mL),
sealed and immersed immediately into the liquid nitrogen
tank. The required number of cryostraws was taken out
from the liquid nitrogen tank and thawed in sufficient
amount of PBS at room temperature. This sperm suspen-
sion was divided into aliquots (1 9 106 cells/mL) and
treated with PBS and 300 lM H2O2 at 4�C for 2 h. These
PBS- and H2O2-treated samples were used as negative and
positive technical controls for both alkaline and neutral
COMET assay. After 2 h of treatment, the technical con-
trols were embedded in agarose gel and subjected to the
lysing procedure along with the other semen samples.
These technical controls were run with each set of samples.
The variations (SD) between 10 sets of samples in tail
intensity were 16.7% for the neutral and 21.0% for the
alkaline assay (running controls). After stimulation with
300 lM H2O2, the corresponding variations were 16.1 and
9.4%, respectively.
During the sampling period in Bandırma, three cryo-
straws were prepared for each single worker. One straw
was stored to use it at Ankara University [Faculty of
Pharmacy, Department of Toxicology (Lab-A)], and the
other straw was stored for use at Hacettepe University
[Faculty of Pharmacy, Department of Toxicology (Lab-
H)]. The last straw was stored as backup. The mean values
of the COMET assay parameters have been compared
between these two laboratories in order to check the
validity of the assay.
After completing the sampling procedure in Bandırma,
all cryostraws were transferred to Lab-A in liquid nitrogen
tank. In order to test the influence of discontinuing the
cooling chain, two cryostraws for each semen sample were
thawed simultaneously in phosphate-buffered saline (PBS)
solution at room temperature and one of them was imme-
diately subjected to COMET assay in Lab-A. The other
semen sample was transferred to Lab-H in PBS solution at
room temperature and subjected to COMET assay. This
procedure led to an interval of 3–5 h between thawing and
start of the COMET assay for the samples analyzed in Lab-
H, resulting in a significant discrepancy of the results.
Therefore, the backup cryostraws were transferred to Lab-
H in liquid nitrogen tank, thawed and immediately re-
analyzed in order to eliminate this potential confounder.
Statistical analysis
Boxplots, Pearson’s correlation coefficient and linear
regressions were used as presented in the figures. For the
data of the COMET assay, a Dunnett test was applied to
compare with one-sided hypotheses the low, medium and
high exposure group with the control group (Bretz et al.
2010). The influences of potential confounders of the
alkaline as well as the neutral tail intensity (such as age,
smoking habit or boron concentration in blood) were
investigated fitting a multiple linear regression model of
the form:
y ¼ b0 þ b1x1 þ b2x2 þ b3x3 þ e ð1Þ
We used a two-sided t-test in order to analyze whether
individual regression coefficients differed significantly from
0 (Sachs 1984). The correlation with other semen parameters
is displayed in Table 1 where the P value represents the
result of a two-sided test with the null hypothesis that the true
correlation coefficient q is equal to 0 (Sachs 1984).
Figures 2 and 3 represent scatter plots of the alkaline
and neutral tail intensity with different independent vari-
ables. Furthermore, each of the plots shows the fitted linear
regression model, the corresponding 95% prediction
interval and Pearson’s correlation coefficient.
All statistical tests were performed with R, a language
and environment for statistical computing, version 2.12.2
(R Development Core Team 2011). The local as well as the
multiple significance levels of the tests were set to 0.05.
Results
The DNA integrity of sperm samples obtained from
workers of the different B exposure groups was assessed by
the tail intensity. These values between the groups were
statistically not different (P [ 0.05) for both the alkaline
and neutral modifications of the COMET assay, as shown
in Fig. 1 (numerical values in Table 1 of the Electronic
Supplementary Material). Therefore, increasing boron
exposures had no additional contribution in addition to
already pre-existing DNA-strand breaks in the sperm cells.
Correlation between mean tail % intensities and boron
concentrations in urine or in semen samples was statisti-
cally not significant (P [ 0.05), while there was a weak but
statistically significant (Pearson, P \ 0.05) correlation
between blood boron concentrations and tail % intensity, in
both the alkaline and neutral assay modifications (Fig. 2).
Correlations between mean tail % intensities and
reproductive toxicity indicators are compiled in Table 1.
30 Arch Toxicol (2012) 86:27–35
123
Statistically significant correlations between mean tail %
intensities and reproductive toxicity indicators were not
observed (P [ 0.05) in alkaline COMET assay. Under
neutral conditions of the COMET assay, the correlations
between mean tail % intensities and some of the repro-
ductive toxicity parameters (motility, motile sperm, normal
morphology, neck/mid-piece defects and tail defects)
reached statistical significance (P \ 0.05; Fig. 3).
Discussion
Several techniques are available to examine the integrity
of sperm DNA. Sperm chromatin structure assay
(SCSA), terminal deoxynucleotidyl transferase-mediated
dUDP nick-end labeling (TUNEL) and single-cell gel
electrophoresis (COMET) assay are commonly used tech-
niques for this purpose. According to recent studies, the
results of the COMET assay are well correlated with those
of the TUNEL and SCSA methods (Aravindan et al. 1997,
Donnelly et al. 2000). This compatibility and the relative
simplicity have led to an increased use of the COMET
assay in this field. There has been discussion about the high
background of DNA damage in sperm in comparison with
somatic cells (Singh et al. 1989). Also, the variability
between studies with the alkaline version is higher than
with the neutral version (Speit et al. 2009). The alkaline
and neutral COMET assay detects both single- and double-
strand breaks in DNA. However, the alkaline conditions
(0.3 M NaOH, pH * 13) also convert alkali-labile sites
into strand breaks. The higher background levels of DNA
migration and higher variability between published studies
Table 1 Correlations between
DNA-strand breaks with semen
parameters and male
reproductive hormone levels
(Duydu et al. 2011)
APV average path velocity,
M/ejac million/ejaculate,
sec. second
Bold values indicate a statistical
significance
Semen parameters Tail % intensity
Alkaline
COMET assay
Neutral
COMET assay
Pearson’s corr. coeff. P value Pearson’s corr. coeff. P value
Motility % 0.017 [0.05 –0.170 <0.05
Motile sperm, M/ejac. –0.075 [0.05 –0.173 <0.05
Sperm conc., M/mL –0.033 [0.05 –0.064 [0.05
All sperm, M/ejac. –0.054 [0.05 –0.130 [0.05
Velocity (APV), lm/sec 0.074 [0.05 –0.118 [0.05
Normal morphology, % 0.020 [0.05 –0.202 <0.01
Head defects, % –0.051 [0.05 –0.096 [0.05
Neck/mid-piece defects, % 0.020 [0.05 0.179 <0.05
Tail defects, % 0.016 [0.05 0.179 <0.05
Cytoplasmic droplets, % 0.091 [0.05 0.042 [0.05
Male reproductive hormones
FSH, mIU/mL 0.024 [0.05 0.034 [0.05
LH, mIU/mL 0.017 [0.05 0.052 [0.05
Testosterone, ng/dl 0.103 [0.05 –0.138 [0.05
HighMediumLowControl
Tail
% in
tens
ity,
alka
line
CO
ME
T
60
50
40
30
20
10
0HighMediumLowControl
Tail
% in
tens
ity,
neut
ral C
OM
ET
10
8
6
4
2
0
Fig. 1 DNA-strand breakage in
sperm cells versus increasing B
blood levels; (o) outliers (*)
extremes, left alkaline COMET
assay, right neutral COMET
assay. Definition of the
exposure groups (Duydu et al.
2011): control group (n = 49,
up to 48.5 ng B/g blood); low
exposure (n = 72, up to 100 ng
B/g blood); medium exposure
(n = 44, up to 150 ng B/g
blood); high exposure group
(n = 39, above 150 ng B/g
blood)
Arch Toxicol (2012) 86:27–35 31
123
with alkaline COMET assay might be a reflection of the
presence of alkali-labile sites in mature human sperm
(Singh et al. 1989). Background values in the neutral ver-
sion are lower than in the alkaline version of the COMET
assay (Singh et al. 1989, Haines et al. 1998, Cordelli et al.
2003). The results of the present study are compatible with
these previous findings.
The inter-individual variability in the amount of DNA-
strand breaks in both control and boron-exposed groups
was remarkable in the alkaline COMET assay. The mean
tail % intensity ranged between 5.0–45.73 and 4.61–53.24
for the control and boron-exposed workers respectively
(Fig. 1). This inter-individual variation makes it difficult to
interpret an external influence on the pre-existing DNA-
strand breaks in sperm cells. The inter-individual variations
are usually attributed to lifestyle factors. Age and smoking
come forward as two potential confounders (Singh et al.
2003, Schmid et al. 2007). The influence of age and
smoking on the tail % intensity in our cohort is shown in
Table 2. Age had a significant impact on the tail %
intensity values in both (alkaline and neutral) COMET
assay versions, while smoking had not. These results were
in agreement with previous data of others (Singh et al.
2003, Schmid et al. 2007).
Although being quite weak, the correlation between
blood boron concentrations and DNA-strand breaks (tail
intensity) of sperm cells reached formal statistical signifi-
cance in both the alkaline (Pearson corr. coeff.: 0.156,
P \ 0.05) and the neutral (Pearson corr. coeff.: -0.184,
P \ 0.05) version of the COMET assay (Fig. 2). However,
the pattern was just opposite between both assay condi-
tions. It was positive under alkaline, but negative under
neutral assay conditions, which does not lend biological
plausibility to this finding. Thus, an adverse effect cannot
be derived.
There is an increasing number of studies on the coher-
ence of sperm DNA integrity and male fertility. High DNA
damage in sperm cells appears as a predictor of infertility
(Sun et al. 1997, Zini et al. 2001, Morris et al. 2002). For
instance, men attending infertility clinics had higher levels
of DNA damage in their sperm (Irvine et al. 2000).
Associations between sperm DNA damage and classical
sperm quality parameters (concentration, morphology, and
motility) have been generally confirmed (Irvine et al. 2000,
Morris et al. 2002).
In our study, there was no statistically significant cor-
relation (P [ 0.05) between the classical sperm parameters
reported previously (Duydu et al. 2011) and DNA-strand
Pearson's corr. coeff.: 0,156, p<0.05
Blood boron, ng/g
0
10
20
30
40
50
Tai
l % in
ten
sity
, alk
alin
e C
OM
ET Pearson's corr. coeff.: 0,126, p>0.05
Urine boron, mg/g creat
10
20
30
40
50
Tai
l % in
ten
sity
, alk
alin
e C
OM
ET Pearson's corr. coeff.: -0,021, p>0.05
Semen boron, ng/g
10
20
30
40
50
Tai
l % n
iten
stiy
, alk
alin
e C
OM
ET
Pearson's corr. coeff.: -0,184, p<0.05
Blood boron, ng/g
0
2
4
6
8
Tai
l % in
ten
sity
, neu
tral
CO
ME
T Pearson's corr. coeff.: -0,088, p>0.05
Urine boron, mg/g creat
0
2
4
6
8
Tai
l % in
ten
sity
, neu
tral
CO
ME
T Pearson's corr. coeff.: 0,021, p>0.05
0 100 200 300 400 0 10 20 30 0 2500 5000 7500
0 100 200 300 400 0 10 20 30 0 2500 5000 7500
Semen boron, ng/g
0
2
4
6
8
Tai
l % in
ten
sity
, neu
tral
CO
ME
T
Fig. 2 Linear regressions with 95% individual prediction intervals.
The correlation between tail % intensity and boron concentrations in
biological fluids is weak (left column: blood boron, middle column:
urine boron, right column: semen boron). However, the correlations
between tail % intensity and blood boron concentrations are
statistically significant (P \ 0.05) in both alkaline (top row) and
neutral (bottom row) conditions of the COMET assay
32 Arch Toxicol (2012) 86:27–35
123
breaks in sperm cells, with the alkaline version of the
COMET assay (Table 1). However, the results of neutral
version of the COMET assay revealed a different picture.
In spite of a weak correlation, the percentage of motile
sperm, the number of motile sperm in ejaculate, the per-
centage of sperm cells with normal morphology, the per-
centage of neck/mid-piece defects and the percentage of
the tail defects were significantly correlated with the
measured DNA-strand breaks in the neutral COMET assay.
The percentage of motile sperm, the number of motile
sperm in the ejaculate and the percentage of sperm cells
with normal morphology were increasing with decreasing
DNA damage, while the percentage of neck/mid-piece
defects and the percentage of the tail defects were
increasing with increasing DNA damage (Fig. 3, Table 1).
Importantly, the DNA-strand breaks of sperm samples
measured in neutral COMET assay were decreasing versus
increasing blood boron concentration (Pearson corr. coeff.:
-0.184, P \ 0.05), which would even point to a ‘‘benefi-
cial’’ effect of B exposure. It was remarkable that signifi-
cant correlations between sperm quality parameters and
mean tail % intensities were observed solely for the results
of neutral COMET assay.
As outlined initially, the first comprehensive study on
possible relations between chronic boron exposure and
human semen parameters was conducted in China (Robbins
et al. 2010, Scialli et al. 2010). Adverse effects of boron on
the semen quality parameters and integrity of sperm DNA
were not reported. The only effect noted was a lower ratio
of Y to X bearing sperm in boron workers compared with
the controls (Robbins et al. 2010). However, the X/Y ratio
was not significantly correlated with semen parameters and
Pearson's corr. coeff.: -0,184, p<0.05
Blood boron, ng/g
0
2
4
6
8
Tai
l % in
ten
sity
, neu
tral
CO
ME
T
Motility %
0
2
4
6
8
Tal
i % in
ten
stiy
, neu
tral
CO
ME
T Pearson's corr. coeff.: -0.170, p<0.05
Motile sperm, M/ejaculate
0
2
4
6
8
Tai
l % in
ten
sity
, neu
tral
CO
ME
T Pearson's corr. coeff.: -0.173, p<0.05
Pearson's corr. coeff.: -0.202, p<0.01
Normal morphology, %
0
2
4
6
8
Tai
l % in
ten
sity
, neu
tral
CO
ME
T
Neck/mid-piece defects, %
0
2
4
6
8T
ail %
inte
nsi
ty, n
eutr
al C
OM
ET Pearson's corr. coeff.: 0.179, p<0.05
0 100 200 300 400 25 50 75 100 0 100 200 300 400 500
0 10 20 30 40 0 10 20 30
Tail defects, %
0
2
4
6
8
Tai
l % in
ten
sity
, neu
tral
CO
ME
T Pearson's corr. coeff.: 0.179, p<0.05
0 10 20 30
Fig. 3 Linear regressions with 95% individual prediction intervals. The correlations between tail % intensity and sperm parameters are weak but
are statistically significant (P \ 0.05) in neutral conditions of the COMET assay
Table 2 Linear regression models for tail % intensity
Input variables Tail % intensity, alkaline COMET
Estimate Std. error P value
Intercept (b0) 32.983 5.729 \0.001
Cigarettes per day (b1) 0.054 0.060 0.374
Age (b2) –0.316 0.132 0.018
Blood boron ng/g (b3) 0.023 0.009 0.016
Input variables Tail % intensity, neutral COMET
Estimate Std. error P value
Intercept (b0) 1.547 0.798 0.054
Cigarettes per day (b1) –0.012 0.008 0.115
Age (b2) 0.038 0.018 0.039
Blood boron ng/g (b3) –0.003 0.001 0.006
Arch Toxicol (2012) 86:27–35 33
123
boron concentrations in biological fluids within each
exposure group (Robbins et al. 2010, Scialli et al. 2010).
The results of our study support the results of the study
conducted in China, with one exception. In the present
study, a negative correlation between blood boron levels
and DNA-strand breaks in neutral COMET assay reached a
weak statistical significance, whereas the correlation
between the same variables was not statistically significant
in the study conducted in China.
In essence, the two recent studies conducted in highly
B-exposed populations in China and in Turkey consistently
arrive at the same conclusion that no adverse effects of
boron on male reproduction can be demonstrated.
Regarding sperm parameters, in both studies, this conclu-
sion is based on classical clinical sperm quality parameters
and on an assessment of DNA integrity using the COMET
assay. The likely reason for these negative results, even in
highly exposed humans, is that the experimental exposures
that had led to reproductive toxicity in animals were sig-
nificantly higher than any B exposures, which are reached
under the conditions of human handling and use (Hubbard
1998, Bolt et al. 2011).
Acknowledgments The project was supported by the National
Boron Research Institute (BOREN) and by the Eti Mine Works
General Management (2008-G0207). The outline of the study and the
processing of the raw data were reviewed by an external expert panel.
The authors thank the reviewers, Professors G. Assennato, Bari, and
M. Guillemin, Lausanne, for their scrutiny and dedicated effort.
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Electronic Supplementary Material
Supplementary Table 1. The integrity of sperm DNA in workers with different B exposures (numerical data to Figure 1).
Exposure groups according to the B blood levels (ng B/g blood) of the workers
COMET version,(Detected parameter)
Control (C)<LOQ
Low exposure (L)>LOQ – 100
Medium exposure (M)>100 – 150
High exposure (H)>150
P values(Dunnet)
Alkaline COMET,(Tail % intensity)
20.35 10.49(5.0 – 45.73)
21.97 10.26(4.61 – 53.24)
23.91 10.35(6.35 – 44.65)
24.62 9.70(7.83 – 46.58) >0.05
Neutral COMET,(Tail % intensity)
2.70 1.38(0.72 – 8.19)
3.10 1.53(0.31 – 8.63)
2.35 1.16(0.26 – 5.62)
2.37 0.86(0.65 – 4.57) >0.05
Mean SD, range in brackets.
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