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: duydu@pharmacy.ankara.edu.tr

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.