Environmental Toxicology and Pharmacology 45 (2016) 68–73

Contents lists available at ScienceDirect

Environmental Toxicology and Pharmacology

journal homepage: www.elsevier.com/locate/etap

Evaluation of urinary metal concentrations and sperm DNA damage in

infertile men from an infertility clinic
Yan Zhou a,∗,1 , Xiao-Ming Fu b,1 , Dong-Liang He c,d , Xue-Min Zou a , Cheng-Qiu Wu c ,
Wei-Zhen Guo a , Wei Feng e
a
School of Public Health, Changsha Medical University, Changsha, Hunan, PR China
b
The 169th Hospital of People’s Liberation Army, Xiangnan Affiliated Hospital of Hunan Normal University, Hengyang, Hunan, PR China
c
Department of Preventive Medicine, School of Public Health, University of South China, Hengyang, Hunan, PR China
d
Department of Nutrition and Food Hygiene, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan,
Hubei, PR China
e
Department of Occupational and Environmental Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology,
Wuhan, Hubei, PR China

a r t i c l e i n f o a b s t r a c t

Article history: This study aimed to examine associations between urinary metal concentrations and sperm DNA damage.
Received 21 December 2015 Thirteen metals [arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), lead
Received in revised form 19 May 2016 (Pb), manganese (Mn), molybdenum (Mo), mercury (Hg), nickel (Ni), selenium (Se), and zinc (Zn)] were
Accepted 21 May 2016
detected in urine samples of 207 infertile men from an infertility clinic using inductively coupled plasma
Available online 24 May 2016
mass spectrometry, and also, sperm DNA damage (tail length, percent DNA tail, and tail distributed
moment) were assessed using neutral comet assay. We found that urinary Hg and Ni were associated
Keywords:
with increasing trends for tail length (both p for trend < 0.05), and that urinary Mn was associated with
Comet assay
Male infertility
increasing trend for tail distributed moment (p for trend = 0.02). These associations did persist even when
Male reproductive health considering multiple metals. Our results suggest that environmental exposure to Hg, Mn, and Ni may be
Metals associated with increased sperm DNA damage.
Sperm DNA damage © 2016 Elsevier B.V. All rights reserved.

1. Introduction als through intake of or contact with these environmental media

A significant increase in the incidence of infertility is an increas-
ing concern in recent years worldwide (Hamilton and Ventura,
2006). It has been reported that male factor infertility contributes
to approximately 50% of infertile couples attending infertility clin-
ics (Agarwal and Said, 2003). Although a number of aetiologies,
including Y-chromosome deletions, gene mutations, aneuploidies,
vas deferens obstruction, varicocele, and erectile dysfunction, have
been identified as potential causes of male infertility (Ollero et al.,
2001), environmental factors such as exposure to certain chemicals
have been also suggested as the potential risks for male infertility
(Sharpe and Irvine, 2004).
Metals, including essential and nonessential elements, are ubiq-
uitously present in various environmental media such as air, water,
soil, dust, and diet. The general population may be exposed to met-
∗ Corresponding author.
E-mail address: nhhdl@126.com (Y. Zhou).
1
These authors equally contributed to this work.
or through supplementation (Meeker et al., 2008). Exposure to
metals has been reported to adversely affect female reproductive
development in toxicological and human studies as reviewed by
Sengupta et al. (2015). Toxicological studies have also shown that
metal exposures have adverse effects on male reproductive health.
Toxic metals such as arsenic (As), cadmium (Cd), lead (Pb), and mer-
cury (Hg) have been shown to disrupt spermatogenesis, decrease
testicular weights, and alter reproductive hormones (Agrawal and
Chansouria, 1989; Moorman et al., 1998; Sarkar et al., 2003; Saygi
et al., 1991). Other metals such as cobalt (Co), chromium (Cr), man-
ganese (Mn), molybdenum (Mo), nickel (Ni), and selenium (Se) are
essential for various biochemical and physiological functions, but
these metals at relatively high levels can also cause adverse male
reproductive outcomes as reviewed by Sengupta (2013).
To date, a larger number of human studies have suggested that
occupational and/or environmental exposure to metals, including
nonessential (e.g., As, Cd, Pb, and Hg) and essential [e.g., cobber
(Cu), Co, Cr, Mo, Mn and Ni] elements, are associated with decreased
conventional semen parameters (e.g., sperm concentration, sperm
motility, and sperm count) and increased sperm nuclear chromatin

http://dx.doi.org/10.1016/j.etap.2016.05.020
1382-6689/© 2016 Elsevier B.V. All rights reserved.
 Y. Zhou et al. / Environmental Toxicology and Pharmacology 45 (2016) 68–73
condensation (Choy et al., 2002; Hernandez-Ochoa et al., 2005;
Kumar et al., 2005; Meeker et al., 2008; Wirth et al., 2007; Xu
et al., 2003; Zeng et al., 2015). However, few studies have exam-
ined the effects of exposure to metals on sperm DNA damage, an
independent and objective marker of sperm function that opposes
to the conventional semen parameters and predicts male fertility
(Agarwal and Said, 2003).
The single-cell gel electrophoresis assay (comet assay) is used
to measure the amount of DNA fragmentation in individual cells.
Comet assay under neutral conditions has been widely used to
assess human sperm DNA damage due to the abundance of alkali-
sensitive sites in sperm (Duty et al., 2002; You et al., 2015).
Increasing evidence suggests that sperm DNA damage adversely
affects male fertility, contributing to poorer embryo development
and lower pregnancy rates among partners of men undergoing
assisted reproductive treatments (Duran et al., 2002; Morris et al.,
2002). In the present study, we examined the association between
urinary metal concentrations and sperm DNA damage using the
neutral comet assay in infertile men from an infertility clinic.
2. Materials and methods
2.1. Study subjects
We recruited study subjects from male partners of sub-fertile
couples who presented to the Reproductive Center of Tongjing
Hospital in Wuhan, China, to seek semen analysis. During March
to May 2012, a total of 252 men agreed to participate in the
study after receiving an explanation about the study purposes.
We excluded 27 azoospermic men, as this may be associated with
an obstructive mechanism or Y-chromosome deletions. We also
excluded 18 men with at least one of the following medical con-
ditions that might alter semen quality: orchiditis, epididymitis,
vesiculitis, vasectomy, undescended testicle, varicocele, injury of
testis, and hernia repair complicated by testicular atrophy. Finally,
a total of 207 men were retained for the current analysis. The sub-
jects at enrollment completed a questionnaire on demographics,
lifestyle habits, occupational exposures, and medical characteris-
tics. Each subject signed a written informed consent, and the study
was approved by the Ethics Committee of Tongji Medical College
(No. [2010]IEC (S032), with a due date of Dec. 31, 2012).
2.2. Semen collection
Before the collection of semen sample, each man was asked to
report his abstinence time. In a dedicated semen collection room
of the hospital, the subject was asked to masturbate into a sterile
plastic specimen container. After liquefaction in a heating chamber
(37 ◦ C) for no more than 60 min, a part of semen sample was used
to analyze the conventional semen parameters. The residual semen
sample was transferred to a 0.5-mL frozen tube and packed into
coolers with ice packs then sent to the laboratory. All the residual
semen samples were stored at liquid nitrogen (−196 ◦ C) until comet
assay analysis. A previous study found no significant differences
in comet assay parameters between the frozen and fresh semen
samples (Duty et al., 2002).
2.3. Neutral comet assay
Sperm DNA damage was measured by using neutral comet assay
according to a previous study, as described in detail elsewhere (You
et al., 2015). Briefly, the frozen semen sample was removed from
the liquid nitrogen and allowed to equilibrate to room temperature.
Ten microliter of semen sample was suspended in 0.7% of low-melt
agarose, and 70-␮L the suspension was added to a slide covered
with normal agarose. Slides were then immersed in a lysis buffer
69
solution for 2.5 h at 4 ◦ C in the dark. After lysis, the slides were
transferred to a solution containing proteinase K and incubated for
3 h at 37 ◦ C. The slides were washed three times with deionized
water at 4 ◦ C and then placed on a horizontal electrophoresis cham-
ber with neutral buffer solution and underwent electrophoresis at
25 V and 300 mA for 20 min. The resulting slides were washed again
with deionized water followed by fixing with ethanol and then air-
dried and stained with 20 ␮g/mL ethidium bromide. Slides were
observed under a fluorescence microscope, and 100 sperm cells
from each slide were randomly selected to measurement. Comet
assay parameters including tail length, percent DNA tail, and tail
distributed moment were evaluated with the Comet Assay Soft-
ware Project Lab (CASP, 2004) image analysis system. The tail length
indicates single-strand breaks of DNA damage, whereas percent
DNA tail and tail distributed moment indicate double-strand breaks
of DNA damage.
2.4. Urine collection and analysis
A single spot urine sample from each subject was collected into
a trace elements-free polyethylene container. After collection, the
urine samples were packed into coolers with ice packs and sent to
the laboratory then stored at −40 ◦ C until analysis. Thirteen met-
als (As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Se, and Zn) were
measured by using inductively coupled plasma mass spectrometry
(ICP-MS), as described in detail elsewhere (Feng et al., 2015). Briefly,
a 3-mL urine sample was acidified with 15-␮L 67% nitric acid solu-
tion (v/v). After digestion, the sample was allowed to equilibrate
to room temperature and 1-mL the sample was further diluted
with 5-mL 1.2% nitric acid solution (v/v), and then was detected
by using an ICP-MS with an octopole based collision/reaction cell.
Standard reference material (2670a) and spiked pooled urine (low
and high concentrations) were also analyzed along with the urine
samples. The spiked recoveries for these metals ranged from 78.3%
to 113.2% and relative standard deviations (RSDs) were not higher
than 10.00%. The limits of detection (LOD) of urinary metals were
as follows: 0.01 ␮g/L for As; 0.002 ␮g/L for Cd; 0.001 ␮g/L for Co;
0.01 ␮g/L for Cr; 0.23 ␮g/L for Cu; 0.29 ␮g/L for Fe; 0.01 ␮g/L for
Hg; 0.02 ␮g/L for Mn; 0.004 ␮g/L for Mo; 0.03 ␮g/L for Ni; 0.18 ␮g/L
for Pb; 0.12 ␮g/L for Se; 0.001 ␮g/L for Zn. Urinary creatinine was
determined with commercial test kits by the picric acid assay to
adjust for the variation in urine diluteness.
2.5. Statistical analysis
We calculated descriptive statistics on demographic character-
istics, urinary metal concentrations, and comet parameters for the
study subjects. We used multivariable linear models to assess dose-
response relationships between quartiles of creatinine-adjusted
urinary metal levels and comet assay parameters. The comet assay
parameters were close to normal distribution and thus were used
in the analysis without any transformation. Covariates including
age, body mass index (BMI), race, smoking status, alcohol use,
education, income, and total tap water consumption were con-
sidered as potential confounders based on statistical and biologic
considerations. The change-in-effect estimate method was used to
determine whether the potential confounders should be included
in the multivariate models. Finally, age and BMI as continuous vari-
ables, smoking status (current and former vs. never smoker) as
dummy variables, and abstinence time as an ordinal five-category
variable (≤2, 3, 4, 5, and ≥6 days) were included in the multivariable
models.
Because certain essential and nonessential metals may addi-
tively, synergistically, or antagonistically affect male reproductive
health (Grotto et al., 2009), we also performed full linear regression
models to simultaneously examine the effects of multiple metals on
70 Y. Zhou et al. / Environmental Toxicology and Pharmacology 45 (2016) 68–73

Table 1 tail length, and tail distributed moment were 26.17%, 55.70 ␮m, and
Demographic characteristics of study subjects (n = 207a ).
7.81 ␮m, respectively.
Characteristics Mean ± standard deviation

Age, years 31.6 ± 5.3 3.3. Associations between urinary metals and comet parameters
Body mass index (BMI), kg/m2 23.7 ± 4.1
Race
Han 202 (97.6) The adjusted associations between quartiles of creatinine-
Other 5 (2.4) adjusted urinary metal levels and comet assay parameters are listed
Abstinence time, days in Fig. 1. We found that most of metals (e.g., As, Cd, Co, Cr, Cu,
≤2 15 (7.2) Fe, Mo, Se, and Zn) were not statistically significantly associated
3 45 (21.7)
4 41 (19.8)
with any of comet assay parameters. However, we found that uri-
5 38 (18.4) nary Hg and Ni were associated with significant increasing trends
≥6 68 (32.9) for tail length (both p for trend < 0.05); men in the fourth quar-
Smoking status tiles of Hg and Ni had significant increases in tail length of 3.14 ␮m
Never 90 (43.7)
(95% CI: 0.70, 5.59) and 2.74 ␮m (95% CI: 0.36, 5.12) compared with
Former 39 (18.9)
Current 77 (37.4) those in the first quartiles, respectively. We found that urinary Mn
Alcohol use was associated with significant increasing trends for tail length (p
Yes 150 (72.5) for trend = 0.04) and tail distributed moment (p for trend = 0.02).
No 57 (27.5) When considering the effects of multiple metals, significant dose-
Education level
Less than high school 81 (39.1)
response relationships between urinary Hg and Ni levels and tail
High school and above 126 (60.9) length and between urinary Mn levels and tail distributed moment
Income, RMB yuan/month were still observed (Table 4). Additionally, we found that men in
≤2000 43 (20.8) the second quartile of Pb had a significant increase in percent tail
2000–6000 137 (66.2)
DNA of 2.33% (95% CI: 0.14, 4.52) compared with those in the first
≥6000 27 (13.0)
Total tap water consumption, mL/day quartile.
<1000 139 (67.5)
≥1000 67 (32.5)
4. Discussion
a
4 missing age, 1 missing smoking status and 1 missing total tap water consump-
tion.
We conducted the first and comprehensive study to estimate
the association between urinary metal concentrations and sperm
sperm DNA damage. We used the backward elimination procedure DNA damage in infertile men from an infertility clinic in China. We
and set alpha at 0.10 for variables to be retained in the final models, found no evidence of associations between most of urinary metals
then added covariates not retained in the final models individu- (e.g., As, Cd, Co, Cr, Cu, Fe, Mo, Se, and Zn) and any of the mea-
ally to further explore evidence of confounding if they changed the sured comet assay parameters. However, we found that urinary
effect estimates for metals by 10% or higher (Meeker et al., 2008; Hg and Ni were associated with increased tail length and urinary
Zeng et al., 2013). We performed the trend test by entering the Mn was associated with increased tail distributed moment. These
quartiles of creatinine-adjusted urinary metal concentrations into associations did persist even when considering the co-exposure of
the model as a continuous ordinal variable using integer values (0, multiple metals. These indicate that environmental exposure to Hg,
1, 2, and 3) (Zeng et al., 2014). We defined statistical significance Ni, and Mn may result in increased sperm DNA damage.
as a p value < 0.05. All the data analysis was performed using the Toxic metals such as As, Cd, Pb, and Hg are the well-known
Statistical Package for the Social Sciences (SPSS), version 18.0 (SPSS reproductive toxicants. A previous animal study showed that expo-
Inc., Chicago, IL, USA). sure to As, Cd, and Pb induced DNA damage in rat primary
spermatocytes (Nava-Hernandez et al., 2009). In this study popu-
3. Results lation, we found that urinary As and Cd were not associated sperm
DNA damage. This discrepancy may be attributed to high doses of
3.1. Demographic characteristics As and Cd used in the animal study. However, we found that the
second quartile of urinary Pb was associated with sperm DNA dam-
The demographic characteristics of the study subjects are listed age. Xu et al. (2003) found that Pb in seminal plasma was associated
in Table 1. Subjects were primarily Han (97.6%) with a mean (±SD) with sperm DNA damage measured as 8-hydroxyl deoxyguanosine
age of 31.6 (±5.3) years and BMI (±SD) of 23.7 (±4.1) kg/m2 . Of the (8-OHdG). Hsu et al. (2009) found that Pb in blood was also associ-
207 study subjects, only 15 (7.2%) men reported their abstinence ated with sperm DNA fragmentation. Exposure to Pb may deplete
time less than two days, 43 (20.8%) men reported their household glutathione (GSH) levels and/or increase the production of reactive
income less than 2000 RMB yuan/month, and 81 (39.1%) had their oxygen species (ROS) that triggers the induction of oxidative stress
education level less than high school. There were 90 (43.7%) never and this status results in sperm DNA damage (Fracasso et al., 2002;
smokers and 57 (27.5%) alcohol users. Xu et al., 2008).
We found that urinary Hg was associated with increased sperm
3.2. Distribution of urinary metal concentrations and comet DNA damage. Hg-induced sperm DNA damage may be triggered by
parameters generating the ROS and altering the antioxidant defense system.
Toxicological and human studies have demonstrated that expo-
The distribution of creatinine-adjusted urinary metal concen- sure to Hg can induce DNA damage by increasing the intracellular
trations is presented in Table 2. All of the measured 13 metals were levels of ROS (Girardi and Elias, 1995). Attacks on DNA by the
detected in 100% of the study subjects with various magnitudes. The ROS, including hydroxyl radical, superoxide anion, singlet oxygen,
highest and lowest geometric mean of creatinine-adjusted urinary and hydrogen peroxide, generally cause oxidative DNA damage in
metal concentrations were Zn (330.93 ␮g/g Cr) and Co (0.15 ␮g/g human sperm (Spano et al., 2000). A previous toxicological study
Cr), respectively. The distribution of comet parameters among the in mice epididymis has shown that exposure to Hg significantly
study subjects is summarized in Table 3. The mean percent tail DNA, decreases glutathione peroxidase (GSH-Px) and GSH levels that
 Y. Zhou et al. / Environmental Toxicology and Pharmacology 45 (2016) 68–73 71

Fig. 1. Adjusted regression coefficients for comet assay parameters associated with quartiles of creatinine-adjusted urinary metal levels, adjusted for age, BMI (continuous),
smoking status (current and former smoker vs. never smoker), and abstinence time (≤2, 3, 4, 5, and ≥6 days).

play an important role in the protection of sperm cells against
oxidative stress (Mahboob et al., 2001).
Mn is one of the essential trace elements required for nor-
mal sperm function (Bansal and Kaur, 2009), but relatively high
levels of Mn have been shown to adversely affect semen quality
in in vitro and in vivo studies (Huang et al., 2001; Ponnapakkam
et al., 2003). Also, a human study has suggested that exposure
to high levels of Mn is associated with decreased sperm motility
and sperm concentration (Wirth et al., 2007). However, there are
no data on Mn-induced DNA damage in human sperm. A previ-
ous study reported that exposure to Mn induced DNA damage in
human peripheral blood lymphocytes as determined by the comet
assay (De Meo et al., 1991). In this study, we found that urinary Mn
was associated with increased sperm DNA damage. The increase in
membrane lipid peroxidation may be a mechanism for Mn-induced
sperm DNA damage. Yiin et al. (1996) have observed that plasma
manganese concentration is significantly associated with the con-
centration of malondialdehyde, the product of lipid peroxidation,
among the manganese workers. Further mechanistic studies are
warranted to confirm the findings.
72 Y. Zhou et al. / Environmental Toxicology and Pharmacology 45 (2016) 68–73

Table 2
Distribution of urinary metal concentrations and comet parameters among the study subjects (n = 207a ).

Metals Mean Geometric mean Selected percentile Range

10th 25th 50th 75th 90th

As 26.58 22.55 11.92 15.40 21.74 30.37 44.39 6.70–246.18

Cd 0.63 0.53 0.27 0.36 0.53 0.79 1.09 0.12–2.28
Co 0.18 0.15 0.08 0.11 0.14 0.20 0.25 0.03–1.25
Cr 1.51 1.11 0.47 0.67 1.07 1.73 2.55 0.17–27.37
Cu 11.25 8.19 4.24 5.22 7.31 10.53 18.87 2.37–249.73
Fe 61.38 35.12 14.93 18.87 28.14 53.46 98.51 8.84–1033.53
Hg 1.17 0.90 0.39 0.54 0.80 1.27 2.51 0.21–10.72
Mn 2.99 1.64 0.65 0.88 1.41 2.72 5.02 0.32–112.51
Mo 47.59 39.34 18.28 26.77 38.94 58.60 85.29 8.43–255.01
Ni 2.29 1.03 0.27 0.60 1.05 1.98 3.13 0.01–91.16
Pb 4.21 3.19 1.51 2.06 2.78 4.37 8.44 0.89–33.67
Se 8.56 7.97 4.98 6.20 7.99 10.25 13.03 2.54–24.01
Zn 410.07 330.93 169.05 236.23 331.01 442.38 726.40 42.47–2838.66
a
2 men were not measured for Fe; 1 men were not measured for Pb; 3 men were not measured for Hg.

Table 3
Distribution of comet parameters among the study subjects (n = 207).

Parameters Mean Geometric mean Selected percentile Range

10th 25th 50th 75th 90th

Percent tail DNA (%) 26.17 25.59 18.81 22.68 26.18 29.49 32.86 13.48–52.98
Tail length (␮m) 55.70 55.38 48.73 51.78 54.86 58.64 65.12 44.44–76.79
Tail distributed moment (␮m) 7.81 7.22 4.52 5.85 7.40 8.95 11.66 1.93–27.07

Table 4 than those reported in a U.S. general population from National

Association of comet assay parameters associated with multiple metals in final linear
Health and Nutrition Examination Survey (NHANES) (0.13, 0.53,
regression models (n = 207).
and 2.08 ␮g/g Cr, respectively), but the geometric mean concen-
Variable ␤-coefficient (95% CI) p for trend trations of Co and Mo (0.15 and 39.34 ␮g/g Cr, respectively) in our
Percent tail DNA (%) study were lower than those reported in the general population
Mn (0.87 and 46.80 ␮g/g Cr, respectively) (Paschal et al., 1998). The geo-
Q1a 0.00 metric mean concentrations of Cd (0.76 ␮g/L) and As (32.41 ␮g/L) in
Q2 0.10 (−2.13, 2.32)
our study were higher than those reported in the adults of the gen-
Q3 3.66 (1.07, 6.24)
Q4 2.96 (−0.37, 6.29) 0.017 eral Flemish population (0.22 ␮g/L and 17.20 ␮g/L, respectively)
Tail length (␮m) (Baeyens et al., 2014). However, the geometric mean concentra-
Hg tions of Cd (0.53 ␮g/g Cr), Co (0.15 ␮g/g Cr), Cr (1.11 ␮g/g Cr), Mn
Q1a 0.00
(1.64 ␮g/g Cr), and Ni (1.03 ␮g/g Cr) in our study were comparable
Q2 1.10 (−1.32, 3.52)
Q3 2.14 (−0.41 4.70) to those reported in the urban adults in Wuhan, China (0.64, 0.18,
Q4 3.72 (1.05, 6.38) 0.005 1.01, 1.84, and 1.72 ␮g/g Cr, respectively) (Feng et al., 2015).
Ni Several limitations needed to be noted in our study. Firstly, as
a
Q1 0.00 with many of the previous human studies, our study was the cross-
Q2 −0.58 (−2.88, 1.71)
sectional design. Therefore, our results are limited in establishing
Q3 −0.36 (−2.71, 1.99)
Q4 2.95 (0.34, 5.56) 0.049 causal relationship between exposure to metals and sperm DNA
Tail distributed moment (␮m) damage. In addition, the men in our study were from an infertility
Mn clinic. Thus, the generalizability of our results to the general popu-
Q1a 0.00
lation is limited. Secondly, we only collected a single urine sample
Q2 0.11 (−1.18, 1.41)
Q3 2.19 (0.72, 3.66)
from each subject to assess the individual’s metal exposure levels.
Q4 1.53 (−0.36, 3.42) 0.017 For longer half-lives of metals, such as Cd, a single measurement
a is likely a reliable biomarker that reflects the body burden over
Reference category.
months or years (Esteban and Castano, 2009). However, for shorter
half-lives of metals, such as As, Mn, and Ni, a single urine sam-
Ni has been shown to be carcinogenic to experimental animals ple may not adequately assess an individual’s exposure category
and/or humans. Exposure to Ni induced oxidative DNA damage in due to the intra-individual variability (Wang et al., 2016). Finally,
somatic tissues has been well characterized (Tkeshelashvili et al., there may be dependent measurement error due to multiple metals
1993). However, the data on Ni-induced DNA damage in human measured in the same urine sample by using the same method, and
sperm are also scarce. In this study, we found that urinary Ni was the small sample size in this study may also result in low statistics
associated with increased sperm DNA damage. Animal studies have power.
shown that exposure to Ni can induce DNA damage in the rat testis
as evidenced by increasing production of ROS (Doreswamy et al., 5. Conclusions
2004). These findings provide an important clue in studying mech-
anism of exposure to Mn on male reproductive toxicity. In the present study, we used urinary metals as biomarkers
The study subjects in this study had a widespread exposure to examine the effects of exposure to metals, including essential
to metals. The geometric mean concentrations of Cr, Mn, and Pb and nonessential elements, on human sperm DNA damage from
in our study (1.11, 1.64, 3.19 ␮g/g Cr, respectively) were higher an infertility clinic. Our results suggest that environmental expo-
 Y. Zhou et al. / Environmental Toxicology and Pharmacology 45 (2016) 68–73
sure to Hg, Mn, and Ni may be associated with increased sperm
DNA damage. However, further human studies with better study
designs, as well as mechanistic studies, are warranted to confirm
the findings.
Conflict of interest
None.
Acknowledgments
We sincerely thank all the study subjects in this study for provid-
ing their urine and semen samples. This work was supported by the
Fund Project of Hunan Province Education Office (Project Number:
2014C0996) and the Fund Project of Hunan Provincial Department
of Health (Project Number: B2013-036).
References
Agarwal, A., Said, T.M., 2003. Role of sperm chromatin abnormalities and DNA
damage in male infertility. Hum. Reprod. Update 9, 331–345.
Agrawal, R., Chansouria, J.P., 1989. Chronic effects of mercuric chloride ingestion
on rat adrenocortical function. Bull Environ. Contam. Toxicol. 43, 481–484.
Baeyens, W., Vrijens, J., Gao, Y., Croes, K., Schoeters, G., Den Hond, E., Sioen, I.,
Bruckers, L., Nawrot, T., Nelen, V., Van den Mieroop, E., Morrens, B., Loots, I.,
Van Larebeke, N., Leermakers, M., 2014. Trace metals in blood and urine of
newborn/mother pairs, adolescents and adults of the Flemish population
(2007–2011). Int. J. Hyg. Environ. Health 217, 878–890.
Bansal, A.K., Kaur, A.R., 2009. Cooperative functions of manganese and thiol redox
system against oxidative stress in human spermatozoa. J. Hum. Reprod. Sci. 2,
76–80.
Choy, C.M., Yeung, Q.S., Briton-Jones, C.M., Cheung, C.K., Lam, C.W., Haines, C.J.,
2002. Relationship between semen parameters and mercury concentrations in
blood and in seminal fluid from subfertile males in Hong Kong. Fertil. Steril. 78,
426–428.
De Meo, M., Laget, M., Castegnaro, M., Dumenil, G., 1991. Genotoxic activity of
potassium permanganate in acidic solutions. Mutat. Res. 260, 295–306.
Doreswamy, K., Shrilatha, B., Rajeshkumar, T., Muralidhara, 2004. Nickel-induced
oxidative stress in testis of mice: evidence of DNA damage and genotoxic
effects. J. Androl. 25, 996–1003.
Duran, E.H., Morshedi, M., Taylor, S., Oehninger, S., 2002. Sperm DNA quality
predicts intrauterine insemination outcome: a prospective cohort study. Hum.
Reprod. 17, 3122–3128.
Duty, S.M., Singh, N.P., Ryan, L., Chen, Z., Lewis, C., Huang, T., Hauser, R., 2002.
Reliability of the comet assay in cryopreserved human sperm. Hum. Reprod.
17, 1274–1280.
Esteban, M., Castano, A., 2009. Non-invasive matrices in human biomonitoring: a
review. Environ. Int. 35, 438–449.
Feng, W., He, X., Chen, M., Deng, S., Qiu, G., Li, X., Liu, C., Li, J., Deng, Q., Huang, S.,
Wang, T., Dai, X., Yang, B., Yuan, J., He, M., Zhang, X., Chen, W., Kan, H., Wu, T.,
2015. Urinary metals and heart rate variability: a cross-sectional study of
urban adults in Wuhan, China. Environ. Health Perspect. 123, 217–222.
Fracasso, M.E., Perbellini, L., Soldà, S., Talamini, G., Franceschetti, P., 2002. Lead
induced DNA strand breaks in lymphocytes of exposed workers: role of
reactive oxygen species and protein kinase C. Mutat. Res. 515, 159–169.
Girardi, G., Elias, M.M., 1995. Mercuric chloride effects on rat renal redox enzymes
activities: SOD protection. Free Radic. Biol. Med. 18, 61–66.
Grotto, D., Barcelos, G.R., Valentini, J., Antunes, L.M., Angeli, J.P., Garcia, S.C.,
Barbosa Jr., F., 2009. Low levels of methylmercury induce DNA damage in rats:
protective effects of selenium. Arch. Toxicol. 83, 249–254.
Hamilton, B.E., Ventura, S.J., 2006. Fertility and abortion rates in the United States
1960–2002. Int. J. Androl. 29, 34–45.
Hernandez-Ochoa, I., Garcia-Vargas, G., Lopez-Carrillo, L., Rubio-Andrade, M.,
Moran-Martinez, J., Cebrian, M.E., Quintanilla-Vega, B., 2005. Low lead
environmental exposure alters semen quality and sperm chromatin
condensation in northern Mexico. Reprod. Toxicol. 20, 221–228.
Hsu, P.C., Chang, H.Y., Guo, Y.L., Liu, Y.C., Shih, T.S., 2009. Effect of smoking on blood
lead levels in workers and role of reactive oxygen species in lead-induced
sperm chromatin DNA damage. Fertil. Steril. 91, 1096–1103.
Huang, Y.L., Tseng, W.C., Lin, T.H., 2001. In vitro effects of metal ions (Fe2+ , Mn2+ ,
Pb2+ ) on sperm motility and lipid peroxidation in human semen. J. Toxicol.
Environ. Health A 62, 259–267.
73
Kumar, S., Sathwara, N.G., Gautam, A.K., Agarwal, K., Shah, B., Kulkarni, P.K., Patel,
K., Patel, A., Dave, L.M., Parikh, D.J., Saiyed, H.N., 2005. Semen quality of
industrial workers occupationally exposed to chromium. J. Occup. Health 47,
424–430.
Mahboob, M., Shireen, K.F., Atkinson, A., Khan, A.T., 2001. Lipid peroxidation and
antioxidant enzyme activity in different organs of mice exposed to low level of
mercury. J. Environ. Sci. Health B 36, 687–697.
Meeker, J.D., Rossano, M.G., Protas, B., Diamond, M.P., Puscheck, E., Daly, D., Paneth,
N., Wirth, J.J., 2008. Cadmium, lead, and other metals in relation to semen
quality: human evidence for molybdenum as a male reproductive toxicant.
Environ. Health Perspect. 116, 1473–1479.
Moorman, W.J., Skaggs, S.R., Clark, J.C., Turner, T.W., Sharpnack, D.D., Murrell, J.A.,
Simon, S.D., Chapin, R.E., Schrader, S.M., 1998. Male reproductive effects of
lead, including species extrapolation for the rabbit model. Reprod. Toxicol. 12,
333–346.
Morris, I.D., Ilott, S., Dixon, L., Brison, D.R., 2002. The spectrum of DNA damage in
human sperm assessed by single cell gel electrophoresis (Comet assay) and its
relationship to fertilization and embryo development. Hum. Reprod. 17,
990–998.
Nava-Hernandez, M.P., Hauad-Marroquin, L.A., Bassol-Mayagoitia, S.,
Garcia-Arenas, G., Mercado-Hernandez, R., Echavarri-Guzman, M.A.,
Cerda-Flores, R.M., 2009. Lead-, cadmium-, and arsenic-induced DNA damage
in rat germinal cells. DNA Cell. Biol. 28, 241–248.
Ollero, M., Gil-Guzman, E., Lopez, M.C., Sharma, R.K., Agarwal, A., Larson, K.,
Evenson, D., Thomas, A.J., Alvarez, J.G., 2001. Characterization of subsets of
human spermatozoa at different stages of maturation: implications in the
diagnosis and treatment of male infertility. Hum. Reprod. 16, 1912–1921.
Paschal, D.C., Ting, B.G., Morrow, J.C., Pirkle, J.L., Jackson, R.J., Sampson, E.J., Miller,
D.T., Caldwell, K.L., 1998. Trace metals in urine of United States residents:
reference range concentrations. Environ. Res. 76, 53–59.
Ponnapakkam, T.P., Bailey, K.S., Graves, K.A., Iszard, M.B., 2003. Assessment of male
reproductive system in the CD-1 mice following oral manganese exposure.
Reprod. Toxicol. 17, 547–551.
Sarkar, M., Chaudhuri, G.R., Chattopadhyay, A., Biswas, N.M., 2003. Effect of sodium
arsenite on spermatogenesis, plasma gonadotrophins and testosterone in rats.
Asian J. Androl. 5, 27–31.
Saygi, S., Deniz, G., Kutsal, O., Vural, N., 1991. Chronic effects of cadmium on
kidney, liver, testis, and fertility of male rats. Biol. Trace Elem. Res. 31, 209–214.
Sengupta, P., Banerjee, R., Nath, S., Das, S., Banerjee, S., 2015. Metals and female
reproductive toxicity. Hum. Exp. Toxicol. 34, 679–697.
Sengupta, P., 2013. Environmental and occupational exposure of metals and their
role in male reproductive functions. Drug Chem. Toxicol. 36, 353–368.
Sharpe, R.M., Irvine, D.S., 2004. How strong is the evidence of a link between
environmental chemicals and adverse effects on human reproductive health?
BMJ 328, 447–451.
Spano, M., Bonde, J.P., Hjollund, H.I., Kolstad, H.A., Cordelli, E., Leter, G., 2000.
Sperm chromatin damage impairs human fertility. The danish first pregnancy
planner study team. Fertil. Steril. 73, 43–50.
Tkeshelashvili, L.K., Reid, T.M., McBride, T.J., Loeb, L.A., 1993. Nickel induces a
signature mutation for oxygen free radical damage. Cancer Res. 53, 4172–4174.
Wang, Y.X., Feng, W., Zeng, Q., Sun, Y., Wang, P., You, L., Yang, P., Huang, Z., Yu, S.L.,
Lu, W.Q., 2016. Variability of metal levels in spot, first morning, and 24-hour
urine samples over a 3-month period in healthy adult Chinese men. Environ.
Health Perspect. 124, 468–476.
Wirth, J.J., Rossano, M.G., Daly, D.C., Paneth, N., Puscheck, E., Potter, R.C., Diamond,
M.P., 2007. Ambient manganese exposure is negatively associated with human
sperm motility and concentration. Epidemiology 18, 270–273.
Xu, D.X., Shen, H.M., Zhu, Q.X., Chua, L., Wang, Q.N., Chia, S.E., Ong, C.N., 2003. The
associations among semen quality, oxidative DNA damage in human
spermatozoa and concentrations of cadmium, lead and selenium in seminal
plasma. Mutat. Res. 534, 155–163.
Xu, J., Lian, L.J., Wu, C., Wang, X.F., Fu, W.Y., Xu, L.H., 2008. Lead induces oxidative
stress, DNA damage and alteration of p53: Bax and Bcl-2 expressions in mice.
Food Chem. Toxicol. 46, 1488–1494.
Yiin, S.J., Lin, T.H., Shih, T.S., 1996. Lipid peroxidation in workers exposed to
manganese. Scand. J. Work Environ. Health 22, 381–386.
You, L., Wang, Y.X., Zeng, Q., Li, M., Huang, Y.H., Hu, Y., Cao, W.C., Liu, A.L., Lu, W.Q.,
2015. Semen phthalate metabolites, spermatozoa apoptosis, and DNA damage:
a cross-sectional study in China. Environ. Sci. Technol. 49, 3805–3812.
Zeng, Q., Zhou, B., Feng, W., Wang, Y.X., Liu, A.L., Yue, J., Li, Y.F., Lu, W.Q., 2013.
Associations of urinary metal concentrations and circulating testosterone in
Chinese men. Reprod. Toxicol. 41, 109–114.
Zeng, Q., Wang, Y.X., Xie, S.H., Xu, L., Chen, Y.Z., Li, M., Yue, J., Li, Y.F., Liu, A.L., Lu,
W.Q., 2014. Drinking-water disinfection by-products and semen quality: a
cross-sectional study in China. Environ. Health Perspect. 122, 741–746.
Zeng, Q., Feng, W., Zhou, B., Wang, Y.X., He, X.S., Yang, P., You, L., Yue, J., Li, Y.F., Lu,
W.Q., 2015. Urinary metal concentrations in relation to semen quality: a
cross-sectional study in China. Environ. Sci. Technol. 49, 5052–5059.