TOXICOLOGICAL SCIENCES 85, 615–623 (2005)
doi:10.1093/toxsci/kfi066
Advance Access publication December 22, 2004
Genotoxic Effects on Spermatozoa of Carbaryl-Exposed Workers
Yankai Xia,* Senping Cheng,* Qian Bian,* Lichun Xu,* Michael D. Collins,† Hebron C. Chang,* Lin Song,*
Jiayin Liu,‡ Shoulin Wang,* and Xinru Wang*,1
*The Key Laboratory of Reproductive Medicine of Jiangsu Province, Institute of Toxicology, Nanjing Medical University, Nanjing 210029, China;
†Department of Environmental Health Sciences, UCLA School of Public Health, Los Angeles, CA 90095; ‡Department of Obstetrics and Gynecology,
the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China
Received August 23, 2004; accepted December 7, 2004
Carbaryl, one of the most important insecticides, is widely
produced and used. To explore carbaryl-induced genotoxic effects
of spermatozoa, particularly DNA damage and chromosome
aberrations (CA), we first examined conventional semen parameters, the progression and motion parameters of the spermatozoa
among 16 carbaryl-exposed workers and 30 internal and external
control individuals. Sperm DNA damage represented as positive
percentage of DNA fragmentation was detected by a modified
terminal deoxy-nucleotidyl transferase-mediated dUTP-biotin
nick end-labeling (TUNEL) assay. Then numerical CA of chromosome X, Y, and 18 were investigated by multicolor fluorescence
in situ hybridization (FISH). The results showed significant
differences in the percentage of sperm abnormality between
carbaryl-exposed group and the external control group (p ¼
0.008). Mean (±SD) percentage of spermatozoa with fragmented
DNA in carbaryl-exposed group (21.04 ± 8.88%) was significantly
higher than those in the internal (13.36 ± 12.17%) and external
control groups (13.92 ± 7.15%), respectively (p ¼ 0.035 and p ¼
0.030). Using FISH, we observed the frequency of sperm sex
chromosome disomy was 0.661 ± 0.238% in the exposed group,
which was significantly higher than that in the external control
group (0.386 ± 0.140%) (p ¼ 0.001), and the carbaryl-exposed
group (0.276 ± 0.126%) had an elevated chromosome 18 disomy
compared with the internal (0.195 ± 0.094%) and external control
groups (0.124 ± 0.068%), respectively (p < 0.05 and p < 0.01). In
addition, carbaryl-exposed donors had significantly higher sperm
nullisomic frequencies of sex chromosomes and chromosome 18
than the external controls (p < 0.01) but not the internal controls.
In summary, the frequencies of aneuploidy and numerical CA
showed significant differences between exposed group and control
groups (p < 0.05 and/or p < 0.01). Moreover, positive correlations
were found between sex chromosome disomy, aneuploidy rate, and
morphologic abnormalities in spermatozoa of all donors (r ¼ 0.564
and r ¼ 0.555, p < 0.01). Our findings suggested that carbaryl
might induce morphologic abnormalities and genotoxic defects of
spermatozoa among exposed workers by causing DNA fragmentation and numerical CA in spermatogenesis as a potential
The authors certify that all research involving human subjects was done under
full compliance with all government policies and the Helsinki Declaration.
1
To whom correspondence should be addressed at Institute of Toxicology,
Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, China.
Fax: þ86-25-86527613. E-mail: [email protected].
genotoxicant. The evidence also indicated that the spermatotoxicity induced by carbaryl exposure might be related to adverse
reproductive outcomes.
Key Words: carbaryl; sperm; genotoxic effect; DNA fragmentation; TUNEL; chromosome aberration; aneuploidy; FISH.
Carbaryl is one of the most important carbamate insecticides
and has been used for over 30 years to control a wide range of
pests, particularly in developing countries. Human carbaryl
exposures from pesticide manufacturing (Baranski, 1993;
Schrag and Dixon, 1985; Wyrobek et al., 1981), crop-dusting
(Savitz et al., 1997) and daily-life contacting (Davis et al.,
1992; Juhler et al., 1999) are common. The toxic effects of
carbaryl related to reproductive toxicology (Baranski, 1993;
Juhler et al., 1999; Savitz et al., 1997; Schrag and Dixon, 1985;
Wyrobek et al., 1981) and genetic toxicology (Delescluse et al.,
2001; Grover et al., 1989; Ishidate and Odashima, 1977; Onfelt
and Klasterska, 1983, 1984; Renglin et al., 1999) have also
been extensively investigated.
Carbaryl-induced genotoxic effects have been reported by in
vitro studies as mitotic aberrations in V79 Chinese hamster
fibroblasts (Renglin et al., 1999) and sister-chromatid exchanges in V79 Chinese hamster cells (Onfelt and Klasterska,
1984). Carbaryl also induced a clastogenic response in some in
vitro bioassays (Ishidate and Odashima, 1977; Onfelt and
Klasterska, 1983). Grover et al. (1989) described carbaryl as a
selective genotoxicant because it could induce both clastogenic
and physiological types of chromosomal aberration. Delescluse
et al. (2001) also suggested that carbaryl provoked a strong
DNA-damaging activity in the human lymphoblastoid cell line.
Previous in vivo studies demonstrated that carbaryl has the
special toxicity to somatic or germ cells in animals (Pant et al.,
1995, 1996; Siboulet et al., 1984), however, others reported the
contrary results (Bigot-Lasserre et al., 2003; Osterloh et al.,
1983). In population-based studies, some epidemiologic and
occupational studies found that carbaryl exposure had correlation with adverse reproductive outcomes such as infertility,
pregnancy loss, and stillbirth (Baranski, 1993; Schrag and
Dixon, 1985). However, the potential mechanisms of these
Toxicological Sciences vol. 85 no. 1 Ó Society of Toxicology 2005; all rights reserved.
616
XIA ET AL.
toxic effects are not clear. Recently, Meeker et al. (2004)
suggested the relationship between carbaryl exposure and
increased DNA damage in human sperm.
Male germ cells are crucial in the reproductive process, and
carbaryl exposure had strong correlation with low semen
quality and sperm shape abnormalities (Juhler et al., 1999;
Wyrobek et al., 1981). Many reports suggested that sperm
DNA damage was related to fertilization and pregnancy
(Benchaib et al., 2003; Carrell et al., 2003a; Henkel et al.,
2004; Sun et al., 1997; Zini et al., 2001). Sperm chromosome
aberrations (CA) were also reported to be associated with
infertility, pregnancy loss, spontaneous abortions, and birth
defects (Carrell et al., 2003b; Hassold and Jacobs, 1984; Shah
et al., 2003). Accordingly, detecting whether carbaryl could
induce spermatotoxic effects in exposed male workers, especially increase sperm CA and DNA damage, is necessary and
helpful to illustrate the possible cause of carbaryl-induced
adverse reproductive outcomes. To date, however, no studies
have focused on these genotoxic effects on spermatozoa of
carbaryl-exposed workers.
Our previous studies reported the sperm genotoxic effects by
pesticide exposure (Bian et al., 2004; Xia et al., 2004).
Recently, our work has provided insight into a variety of
genotoxic effects on spermatozoa by which carbaryl exposure
could induce adverse reproductive outcomes. In this study we
first investigated the conventional semen parameters among
donors from the carbaryl-exposed group and the internal and
external control groups according to WHO Laboratory Manual
for the Examination of Human Semen and Sperm-Cervical
Mucus Interaction (World Health Organization, 1999). The
progression and motion parameters were assessed by using
computer-assisted sperm analysis (CASA). Then terminal
deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin
nick end-labeling (TUNEL) assay was used to evaluate sperm
DNA fragmentation. And due to fluorescence in situ hybridization (FISH) becoming the efficient method to evaluate the
genotoxic effects of environmental and occupational factors on
male gametes (Padungtod et al., 1999; Xu et al., 2003), we
performed multicolor FISH to examine numerical CA by using
the centromeric DNA probes of sex chromosomes and
chromosome 18, since the numerical CA of these chromosomes were familiar in newborns or infertile men and some
chemicals could induce increased numerical CA of these
chromosomes (De Mas et al., 2001; Padungtod et al., 1999).
MATERIALS AND METHODS
Study population. This study was conducted in Changzhou, China, and 46
sperm donors aged from 21 to 48 years were included. For human sperm,
donors were healthy, young, nonsmokers and nonregular drinkers. Sixteen of
them were carbaryl-exposed workers, who had been worked in the plant for
over 1 year and had been working continuously for 6 months before biological
sampling. They were randomly selected from the same working area. Another
12 internal control individuals were clerical or official workers who were in the
same pesticide factory but far away from the pesticide workshop. Eighteen
persons in the external control group were selected from the professions other
than pesticide workers. These subjects had no history of exposures to carbaryl
or other genotoxic chemicals. All the subjects provided their written informed
consent and completed a face-to-face questionnaire concerning standard
demographic data as well as medication, lifestyle, and occupational exposure.
It was ensured that carbaryl-exposed workers and the control cohorts did not
markedly differ from each other except for occupational exposure. All the
subjects were paid for their participation. The protocol and consent form, which
the subjects read and signed, were approved by the ethical committee of
Nanjing Medical University, and an IRB (Institutional Review Board) approval
was given prior to this study.
Carbaryl-exposed donors were recruited from the same chemical plant,
reported no chronic diseases or genetic syndromes (Klinefelter, Edwards, XYY,
syndromes etc.), and had no previous exposure to chemotherapy or radiotherapy.
Data and biological specimens collections. We used CD-I air sampler
(Beijing Detection Instrument Factory, Beijing, China) to detect the air
concentration of carbaryl at different working areas of three groups for 3 days
continuously. At the end of the work shift once, we conducted exposure
assessment on three randomly selected subjects per day for 3 days. This
assessment consisted of two components, the individual sampling by using
active personal sampler (Xinyu Analysis Instrument Factory, Jiangsu, China)
and sampling of dermal contamination by attaching fibrous filter membrane to
ten body areas. In the workplace, the mean air concentration of carbaryl was
41.19 3 103 mg/m3, which was significantly higher than those in the internal
control area (6.30 3 103 mg/m3, p < 0.01) and external control area
(undetected). Simultaneously, the mean concentrations expressed as a timeweighted average of carbaryl with the individual sampling (7.38 mg/m3) and
the dermal contamination (862.47 mg/m2) detected in the carbaryl exposure
area were significantly higher than those in control areas (undetected). The
actual exposure of carbaryl with the individual sampling in the exposure area
was higher than 5 mg/m3, the permissible exposure limits set by ACGIH and
OSHA (American Conference of Governmental Industrial Hygienists, 1999;
Occupational Safety and Health Administration, 1998).
We performed the preliminary evaluation on health status of workers
including the general situation, the cardiovascular system (blood pressure and
cardiogram), the nerve system, and the reproductive system. No significant
differences between carbaryl-exposed group and control groups were found.
Semen analysis. All semen samples were obtained in a private room by
masturbation into a sterile wide-mouth and metal-free plastic container after
a recommended 3-day sexual abstinence.
After liquefaction at 37°C for 30 min and within 1 h of production, we
performed conventional semen analysis according to WHO guidelines, including semen volume, sperm concentration, sperm number per ejaculum,
sperm motility, and sperm morphologic abnormalities, by using a light
microscopy (LABOPHOT-2, Nikon) and Micro-cell slide. To assess the
morphology of spermatozoa, semen smear was prepared on a slide and airdried. These smears were fixed and stained according to WHO guidelines
(World Health Organization, 1999). The sperm morphologic abnormalities can
be classified into four categories: head abnormality, neck/mid-piece abnormality, tail abnormality, and mixed abnormality. The sperm progression and motion
parameters were evaluated by CASA (Hobson Sperm Tracker, UK, software
HST-7V1B; settings parameters included search radius, 28.88 mm; trail, 59;
thresholds, þ18/100; p.win, 0.8 s; refresh time, 2 s). These parameters were
determined for sperm tracts: beat cross frequency (BCF, Hz), amplitude of lateral
head displacement (ALH, mm), linearity (LIN, %), straightness (STR, %),
average path velocity (VAP, mm/s), curvilinear velocity (VCL, mm/s), and
straight-line velocity (VSL, mm/s). Strict quality control measures were enforced throughout the study. Each sample must be detected twice successively.
Observation and counting in the semen analysis were processed by different
technicians, and the background of samples were blinded to avoid bias.
Semen samples were allocated into several Eppendorf tubes and stored at
70°C for subsequent experiments.
617
GENOTOXIC EFFECTS ON SPERMATOZOA
TUNEL assay. A FITC-labeled dUTP system (in-situ cell death detection
kit, Fluorescein, Roche, Germany) was applied to measure sperm DNA
fragmentation.
The samples were thawed in a 37°C water bath and were washed twice
(1500 g, 5 min) in Ca2þ- and Mg2þ-free phosphate-buffered saline (PBS) at
4°C. Sperm suspension with appropriate concentration (about 3 3 106
spermatozoa per sample) was fixed in 2% paraformaldehyde (pH 7.4) for
30 min at room temperature (RT). Fixed cells were resuspended in 100 ll
permeabilisation solution (0.1% Triton X-100, 0.1% sodium citrate) for 10 min
on ice after washed with PBS again at 4°C. Then the samples were washed with
PBS once, and cells were resuspended in 50 ll TdT reaction solution containing
nucleotides and TdT enzyme. One tube of control sample was kept as a negative
control without enzyme addition. The samples were incubated in a humidified
chamber for 60 min at 37°C in the dark. At the end of incubation, samples were
centrifuged, and cells were resuspended in PBS for flow cytometric (FCM)
analysis after the reaction solution was discarded. The samples were analyzed
using FCM with an air-cooled argon 488 nm laser and a 550 nm dichroic mirror
as detectors. At least 10,000 cells were collected in each group. The obtained
data were finally analyzed by Cell Quest software (Version 3.2.1, Becton
Dickinson Immunocytometry Systems, Silicon Valley, CA) for calculating the
percentage of FITC-labeled dUTP-positive cells.
By using DNase I (Sigma, St. Louis, MO) as the positive control, we found
that treatment of DNase I with sperm cells resulted in a significant increase of
spermatozoa labeled with FITC.
Multicolor FISH. We performed multicolor FISH in decondensed sperm
nuclei of samples from 46 donors by using DNA probes specific for the
centromeric regions of sex chromosomes and chromosome 18.
All of the samples were thawed at RT and washed three times (800 g, 10 min,
4°C) in 0.01 M Tris-0.09% NaCl buffer (pH 8.0) at 4°C. Sperm suspension with
appropriate concentration was smeared onto a 3-cm2 area of an ethanol-cleaned
microscope slide and was air-dried for 2 days at RT. Firstly, sperm nuclei were
immerged into PBS/0.1% Tween-20 for 30 min. After rinsed by PBS twice, the
slides were decondensed in 10 mM dithiothreitol (DTT, pH 8.0) solution for 2 h
at RT and rinsed in 23 saline-sodium citrate buffer (SSC, Qbiogene, Montreal,
Canada) twice. Then we used RNase A (Sigma, St. Louis, MO) and proteinase
K (Sigma, St. Louis, MO) to remove RNA and digest the protein in sperm cell
nuclei. After being dehydrated in 70, 80, and 100% ethanol at RT for 2 min each
and immersed into 23 SSC at 37°C for 30 min, sperm slides were denatured in
a solution of 70% formamide/23 SSC at 72°C for 3 min, then snap cooled and
dehydrated in 70, 80, and 100% ethanol at 20°C for 2 min each, air-dried and
prewarmed to 37°C. At the same time, probe mix was prepared by mixing 5 ll
of D18Z1 probe (chromosome 18 satellite probe, direct blue, Qbiogene,
Montreal, Canada) and 5 ll of DXZ1/DYZ3 probe (chromosome X/Y cocktail
probe, direct labeled, Xcen (DXZ1) green, Ycen (DYZ3) red, Qbiogene,
Montreal, Canada); then the probes were mixed and heated at 96°C for 10 min
and 5 min, respectively. After being denatured, probes were chilled on ice for
10 min. Ten microliters of probe mix was pipetted onto the slide area over the
sperm. The slide was coverslipped, sealed with sealing glue, and incubated in
a humidified chamber at 37°C for over 16 h in the dark. We rinsed the slides in
wash buffer (0.53 SSC/0.1%SDS) at 65°C and 13 phosphate-buffered
detergent (PBD, Qbiogene, Montreal, Canada) at RT for 5 min each. The
slides were counterstained by 10 ll DAPI/Antifade (Qbiogene, Montreal,
Canada) with glass coverslip. Finally, we viewed the slides under a fluorescence
microscope (Nikon E400) equipped with DAPI/Rhodamine/FITC/DEAC fourfold band filter set with 10003 magnification.
Observation and counting were processed by three different persons who
were blinded to the exposure status of the donors by using Leica QFISH
software (Version V2.3a, Leica Imaging Systems, Cambridge, UK). Slides
were used for counting only when the hybridization efficiency exceeded 98%.
About 10,000 sperm nuclei were counted for each sample. Stringent criteria
were applied during counting: the signals had to be of equal intensity,
comparable brightness and size, be separated from each other, be regular in
appearance, not diffuse, and clearly positioned within the sperm head.
Overlapping nuclei, disrupted nuclei with indistinct margins, very large nuclei
FIG. 1. Inter-technician differences on the counting percentage of sperm
aneuploidy. Carl, Car2, and Car3: percentage of sperm aneuploidy of carbarylexposed group evaluated by technician 1, technician 2, and technician 3,
respectively; E-conl, E-con2, and E-con3: percentage of sperm aneuploidy of
the external control group evaluated by technician 1, technician 2, and
technician 3, respectively; I-conl, I-con2, and I-con3: percentage of sperm
aneuploidy of the internal control group evaluated by technician 1, technician 2,
and technician 3, respectively.
with diffused chromatin, and very small nuclei with no signals of spermatozoa
were eliminated from scoring.
Statistical analysis. We performed one-way ANOVA to compare the
differences of conventional semen parameters, CASA results, DNA damage
profiles, and numerical CA between carbaryl-exposed group and control groups
by the SPSS for Windows (Version 10.0). Dunnett-test or Dunnett’s C-test were
used as appropriate. Statistical significance was assumed to p 0.05. Bivariate
correlation analysis was used for detecting correlation among the parameters of
numerical CA, DNA damage, and conventional semen analysis. Due to
avoiding the effects of some confounding factors such as smoking and alcohol
consumption, age became the most likely confounding factor for the relationship between carbaryl exposure and sperm genotoxic effects. For this study, age
was restricted as an eligibility criterion and showed no evidence of differences
among three groups.
Since there were three technicians evaluating sperm slides repetitively, we
also investigated the inter-technician differences. Descriptive statistics shown
in Figure 1 revealed that each technician scored similar numbers of aneuploidy,
though there was a weak tendency for a technician to score higher than others in
each subject.
RESULTS
Study Population
The age and work years of the donors showed no significant
differences between carbaryl-exposed group and control
groups (Table 1).
Semen Analysis
Conventional semen analysis showed no significant differences in semen volume, sperm concentration, sperm number
per ejaculum, and sperm motility between carbaryl-exposed
618
XIA ET AL.
TABLE 1
Characteristics and Conventional Semen Parameters of the
Study Populationa
TABLE 2
Comparison of Computer Assisted Sperm Analysis Profiles
Between Carbaryl-Exposed Group and Control Groups
Controls (mean ± SD)
Characteristics and
semen parameters
Carbaryl-exposed
(mean ± SD)
(n ¼ 16)
Age (years)
Work years (years)
Semen parameters
Semen volume (ml)
Sperm concentration
(3106/ml)
Sperm number per
ejaculum (3106)
Sperm motility
(% motile)
Sperm abnormalities (%)
Head abnormality (%)
Neck/mid-piece
abnormality (%)
Tail abnormality (%)
Mixed abnormality (%)
28.23 ± 9.22
7.88 ± 7.78
Internal
controls
(n ¼ 16)
External
controls
(n ¼ 16)
28.62 ± 7.93 30.10 ± 5.41
6.21 ± 5.45
2.82 ± 1.45
2.88 ± 1.49 2.77 ± 1.34
50.10 ± 24.53 45.15 ± 26.13 49.18 ± 27.62
Controls (mean ± SD)
Carbaryl-exposed
Internals controls External controls
CASA profiles (mean ± SD) (n ¼ 16)
(n ¼ 12)
(n ¼ 18)
BCF (Hz)
ALH (lm)
LIN (%)
STR(%)
VAP (lm/sec)
VCL (lm/sec)
VSL (lm/sec)
4.76
10.71
43.21
75.34
40.24
74.26
31.09
±
±
±
±
±
±
±
1.74
2.86
8.96
11.45
7.36
20.10
9.71
4.55
11.43
43.62
78.13
38.82
71.90
30.22
±
±
±
±
±
±
±
0.98
1.97
8.01
6.56
5.27
13.57
6.40
4.73
11.21
45.00
79.81
38.86
71.32
30.72
±
±
±
±
±
±
±
1.48
2.41
6.66
5.56
6.78
17.31
8.09
135.38 ± 81.49 101.62 ± 81.27111.17 ± 86.04
60.38 ± 13.02 57.41 ± 9.72 57.02 ± 14.52
25.25 ± 4.90c
11.72 ± 4.93b
1.88 ± 1.32
10.82 ± 3.09c
0.83 ± 0.55
Note. BCF, beat cross frequency; ALH, amplitude of lateral head
displacement; LIN, linearity; STR, straightness; VAP, average path velocity;
VCL, curvilinear velocity; VSL, straight line velocity. n ¼ 46.
20.50 ± 6.71 15.92 ± 7.58
9.85 ± 5.12 7.56 ± 3.61
1.19 ± 0.94 1.18 ± 1.03
8.73 ± 4.10
0.72 ± 0.62
6.60 ± 4.78
0.58 ± 0.56
a
n ¼ 46.
p < 0.05 compared with external controls.
c
p < 0.01 compared with external controls.
b
group and control groups. However, the morphological defects
as sperm abnormalities (including head abnormality, neck/midpiece abnormality, tail abnormality, and mixed abnormality) in
the carbaryl-exposed group were significantly higher than
those in the external control group (p ¼ 0.008) (Table 1).
From a CASA study, we detected that, in carbaryl-exposed
group, the progression and motion parameters such as beat
cross frequency (BCF), amplitude of lateral head displacement
(ALH), linearity (LIN), straightness (STR), average path
velocity (VAP), curvilinear velocity (VCL), and straight line
velocity (VSL) had no significant differences compared with
control groups (Table 2).
spermatozoa from semen samples of 46 donors in total. The
overall hybridization efficiency was 99.32%.
The average X:Y ratio of chromosomes in the whole study
groups was 1.002 ± 0.085, and no significant differences were
observed in the percentage of normal spermatozoa containing
X chromosome or Y chromosome between any two groups.
There were no significant differences between carbarylexposed workers and control individuals with respect to the
diploidy rates (Table 3).
We observed significant differences in the frequencies of
disomic sex-chromosome-bearing (highest the XY-bearing)
and disomic chromosome 18 spermatozoa between carbarylexposed group and control groups, respectively (p < 0.05 and/
or p < 0.01). We also found the nullisomies of sex chromosomes and chromosome 18 were significantly higher than those
TUNEL Assay
DNA fragmentation was assessed among 46 donors by using
a TUNEL assay. Less than 4% of cells in the negative control
sample showed signals, and more than 96% of cells in positive
control samples showed signals. Our results showed the mean
(±SD) percentage of spermatozoa with fragmented DNA in the
carbaryl-exposed group (21.04 ± 8.88%) was significantly
higher than those in the internal (13.36 ± 12.17%) and external
control groups (13.92 ± 7.15%), respectively (p ¼ 0.035 and
p ¼ 0.030) (Fig. 2).
Multicolor FISH
We used a multicolor FISH assay to detect numerical CA in
sex chromosomes and chromosome 18. We counted 466,866
FIG. 2. Comparison of positive percentage of sperm DNA fragmentation
between carbaryl-exposed group and the internal and external control groups.
*p ¼ 0.035 compared with internal controls, #p ¼ 0.030 compared with
external controls.
619
GENOTOXIC EFFECTS ON SPERMATOZOA
TABLE 3
Comparison of Frequencies of Numerical Chromosome
Aberrations Between Carbaryl-Exposed Group and
Control Groups
Controls (mean ± SD)
FISH results
Counted sperm
Normal sperm (%)
X/Y
Diploid (%)
XX1818
XY1818
YY1818
sum
Disomic sperm (%)
XX18
XY18
YY18
d-sex
X1818
Y1818
d-18
Nullisomic sperm (%)
n-sex
n-18
Carbaryl-exposed
(mean ± SD)
(n ¼ 16)
Internal
controls
(n ¼ 12)
External
controls
(n ¼ 18)
160268
156500 (97.65)
1.009 ± 0.086
122335
184263
120168 (98.23) 181384 (98.44)
0.999 ± 0.084 0.998 ± 0.090
0.022
0.034
0.027
0.083
±
±
±
±
0.020
0.024
0.012
0.035
0.018
0.020
0.016
0.054
±
±
±
±
0.014
0.021
0.023
0.047
0.018
0.037
0.026
0.081
±
±
±
±
0.017
0.026
0.018
0.044
0.150
0.281
0.185
0.661
0.113
0.119
0.276
±
±
±
±
±
±
±
0.097
0.102c
0.083a,c
0.238c
0.070b
0.055a,c
0.126a,c
0.085
0.280
0.134
0.563
0.093
0.076
0.195
±
±
±
±
±
±
±
0.052
0.076
0.052
0.135
0.053
0.031
0.094
0.083
0.177
0.079
0.386
0.051
0.052
0.124
±
±
±
±
±
±
±
0.042
0.080
0.042
0.140
0.028
0.043
0.068
0.426 ± 0.174c
0.268 ± 0.126a
0.383 ± 0.099
0.222 ± 0.062
0.227 ± 0.077
0.139 ± 0.043
Note. d-sex, sex chromosome disomy; d-18, chromosome 18 disomy; n-sex,
sex chromosome nullisomy; n-18, chromosome 18 nullisomy. n ¼ 46.
a
p < 0.05 compared with internal controls.
b
p < 0.05 compared with external controls.
c
p < 0.01 compared with external controls.
in the external controls (p < 0.01) but not the internal controls,
as shown in Figure 3 and Table 3. Moreover, the frequencies of
total aneuploidy and numerical CA (aneuploidy including
disomy and nullisomy of sex chromosomes and chromosome
18; numerical CA including aneuploidy and diploidy of sex
chromosomes and chromosome 18) showed significant differences between carbaryl-exposed group and control groups (p <
0.05 and/or p < 0.01) (Fig. 4).
In addition, we compared the frequencies of nullisomy and
disomy. The results showed the strong correlation between
disomic and nullisomic frequencies of sex chromosomes and
chromosome 18 (r > 0.70, p < 0.001). Positive correlation was
also found between the frequencies of spermatozoa with
chromosome 18 and sex chromosome aneuploidies (r > 0.63,
p < 0.001).
Correlation analyses showed the positive correlation not
only between disomic and nullisomic frequencies of these
chromosomes, aneuploidy rate, and numerical CA rate (r >
0.80, p < 0.001), but also between sex chromosome disomy,
aneuploidy rate, and sperm abnormality in spermatozoa of all
donors (r ¼ 0.564 and r ¼ 0.555, p < 0.01).
DISCUSSION
A number of agricultural chemicals, including carbaryl
(Baranski, 1993; Schrag and Dixon, 1985) affect the reproductive system, resulting in adverse outcomes such as abortion,
stillbirth, birth defects, and infertility. However, the relationships between occupational and environmental chemicals and
these outcomes remain poorly defined. Therefore, it is essential
to understand how and what outcomes arise from carbaryl
exposure.
Owing to their pivotal effects in the reproductive process,
germ cells, especially spermatozoa, have been of interest, with
studies of chemical exposure in relation to poor semen quality
and genotoxic effects on spermatozoa resulting in adverse
reproductive endpoints (Oliva et al., 2001; Naccarati et al.,
2003; Schrag and Dixon, 1985). As we know, sperm genotoxic
effects such as increased sperm DNA damage and CA are
important reasons for these endpoints to occur (Benchaib et al.,
2003, Carrell et al., 2003a,b; Hassold and Jacobs, 1984; Henkel
et al., 2004; Zini et al., 2001). Thus, we conducted this study to
detect the relationship between carbaryl exposure and its sperm
genotoxic effects, and to illustrate the possible mechani sms in
the process of inducing adverse reproductive outcomes by
carbaryl.
First of all, we found some conventional semen parameters
were not related to carbaryl exposure. These results were
consistent with the former article, in which there was an
elevated level of sperm with an extra Y chromosome by a FISH
analysis, while other semen parameters remained unchanged
(Selevan et al., 2000). The CASA profiles also showed no
significant differences between exposed group and control
groups. However, the percentage of sperm abnormality in
carbaryl-exposed group was significantly higher than that in the
external control group (p ¼ 0.008). There was also positive
correlation between sex chromosome disomy, aneuploidy rate,
and sperm abnormality (r ¼ 0.564 and r ¼ 0.555, p < 0.01).
The abnormal spermatozoa may be attended by severe CA, and
the frequency of abnormality may be related to aneuploidy. Our
results were consistent with the reports by Monosson et al.
(1999), Vicari et al. (2003) and Xia et al. (2004). Styrna et al.
(2003) also suggested the relationship between sperm morphologic abnormality and Y chromosome deletion.
Currently, a variety of biomarkers are used to assess the
potential adverse reproductive effects due to toxic chemical
exposures. Many reports suggested that sperm DNA damage
represented as fragmentation was associated with lower
pregnancy rates, recurrent pregnancy loss, fertilization rate,
infertility, and genetic disease in the offspring (Carrell et al.,
2003a; Henkel et al., 2004; Sun et al., 1997; Zini et al., 2001).
Sperm with DNA fragmentation can still fertilize an oocyte, but
when paternal genes are switched on, further embryonic
development stops, resulting in failed pregnancy. Henkel
et al. (2004) suggested that DNA fragmentation in human
sperm could be caused by external factors, such as reactive
620
XIA ET AL.
FIG. 3. Photomicrographs of spermatozoa by multicolor FISH (green signal: X chromosome labeled by FITC; red signal: Y chromosome labeled by
Rhodamine; blue signal: chromosome 18 labeled by DEAC). (A) Photomicrographs of spermatozoa by multicolor FISH (10003 magnification); (B–C) normal
sperm (X18 and Y18); (D–F) diploid sperm (XX1818, XY1818, and YY1818); (G–I) disomic sex chromosome sperm (XX18, XY18, and YY18); (J–K) disomic
chromosome 18 sperm (X1818 and Y1818); (L–M) nullisomic sperm (X and Y).
oxygen species, rather than by apoptosis. Exposure to known
genotoxic compounds could induce DNA damage directly or
through other mechanisms, such as oxidative stress or inflammatory processes (Lebailly et al., 1998). More recently,
Meeker et al. (2004) suggested that environmental exposure to
carbaryl may be associated with increased DNA damage in
human sperm.
FIG. 4. Comparison of percentages of sperm aneuploidy and numerical
chromosome aberrations between carbaryl-exposed group and the internal and
external control groups (aneuploidy including disomy and nullisomy of sex
chromosomes and chromosome 18; numerical chromosome aberrations including aneuploidy and diploidy of sex chromosomes and chromosome 18). *p <
0.05 compared with internal controls, ##p < 0.01 compared with external
controls.
TUNEL assay was originally designed for measuring DNA
fragmentation during apoptosis (Gavrieli et al., 1992). However, this analysis is not possible for spermatozoa, due to the
presence of protamines in sperm. By using the FCM TUNEL
assay, we can detect DNA fragments of damage spermatozoa.
Some reports suggested that DNA fragmentation, as determined by the TUNEL assay, is predictive for pregnancy
(Henkel et al., 2004), intracytoplasmic sperm injection (ICSI)
outcome (Benchaib et al., 2003), and late paternal effect
(Tesarik et al., 2004).
Our findings demonstrated that, for carbaryl-exposed workers, the proportion of sperm showing DNA fragmentation was
significantly higher than those in control groups. Though the
prevalence of DNA fragmentation in human sperm closely
correlated with semen parameters such as sperm concentration,
motility, and morphology (Benchaib et al., 2003; Muratori
et al., 2000; Sun et al., 1997; Zini et al., 2001), our results did
not show any significant relationships between percentage of
DNA fragmentation and conventional semen parameters or
CASA profiles. Therefore, it appeared that sperm DNA
fragmentation, which may be indicative of the early stages of
damage, was detectable by using a TUNEL assay when there
were no other indications such as significant changes in sperm
motility and concentration could be applied.
Chromosome abnormalities, the majority of which are
paternally derived, can lead to abnormal reproductive outcomes, in which the most common is early loss of the
GENOTOXIC EFFECTS ON SPERMATOZOA
pregnancy, as well as genetic diseases in offspring. Of lost
conceptions, over 50% carry chromosomal defects, including
aneuploidies and structural aberrations (McFadden and
Friedman, 1997). It is important to address the issue of sperm
CA because mitosis of spermatogonia and meiosis of spermatocytes occur throughout adult life, and these processes may
be susceptible to the effects of environmental exposures. Men
exposed to genotoxic agents showed elevated frequencies of
spermatozoa with CA (Harkonen et al., 1999; Naccarati et al.,
2003; Robbins et al., 1997; Sram et al., 1999; Xu et al., 2003).
However, no reports on the association between CA and
carbaryl exposure in human germ cells are available. In this
study, we evaluated the possible association between carbaryl
exposure and numerical CA in human sperm by a multicolor
FISH assay. Our results showed that carbaryl exposure was not
associated with imbalance of the X:Y ratio. There were also no
significant differences between carbaryl-exposed workers and
control individuals with respect to the diploidy.
Aneuploidy, the primary abnormality of chromosome number, is the most prevalent type of genetic abnormality in human.
Based on conservative estimates, aneuploidy in the general
population could be around 7% (Vidal et al., 2001). Aneuploidy in germ cells is the major cause of infertility, abortion,
and congenital diseases, and is widely suggested to be a leading
cause of spontaneous abortions (Nishikawa et al., 2000). A
substantial proportion of aneuploidy occurring in embryos and
new-borns is of paternal origin (Tang et al., 2004), but little is
known about the causes of aneuploidy in human sperm,
particularly the contribution of environmental and occupational
exposures such as pesticide exposure. As we know, varied
bioactive compounds of the environment are capable of
crossing the blood–testis barrier and affecting male reproductive mechanisms (Okumura et al., 1975). Different exogenous
agents may interfere with the normal disjunction of sister
chromosomes during meiosis and increase the frequency of
aneuploid sperm, as demonstrated in mice treated with
aneugens (Giri et al., 2002) and men treated with anticancer
agents (Robbins et al., 1997). Although not all germ cells
achieve maturity (Braun, 1998), the close correlation of
chromosome nondisjunction in spermatogenesis with genotoxic outcomes indicates that the investigation for aneuploidy
in a large sample of aneugen-exposed workers is significant.
Among aneuploidies, sex chromosome aneuploidies (e.g., XO,
XXY, XYY) have a substantial paternal contribution. About
0–17% autosomal aneuploidies come from agnate, and the
higher rate appears in sex chromosomal aneuploidies.
According to these reports, there was an association between
male infertility and embryo with aneuploidy of sex chromosomes (Nishikawa et al., 2000), and the fraction of affected
offspring in which the extra chromosome is of paternal origin is
estimated to be 44% for 47,XXY and 100% for 47,XYY
(Abruzzo and Hossold, 1995). Among 45,X cases, about 77%
of newborns and 83% of spontaneous abortions are due to lack
of paternal chromosome (Hassold et al., 1992). Our observa-
621
tions suggested that carbaryl exposure might induce sex
chromosome nondisjunction in spermatogenesis, and this sex
chromosome aneuploidy might be related to adverse reproductive outcomes among family members of carbaryl-exposed
workers. Due to the young age of the carbaryl-exposed workers
in our study, only six were married, and one of their wives had
spontaneous abortion once. This outcome may be a coincidence, and further studies should be required to assess the
prevalence of spontaneous abortions, birth defects, and genetic
syndromes of their offspring.
In conclusion, the results presented here provided evidences
of an important relationship between occupational carbaryl
exposure and sperm genotoxic effects. Moreover, an elevated
level of morphologic defects was detected in spermatozoa of
carbaryl-exposed workers compared with control cohorts.
However, there was a lack of striking associations between
carbaryl exposure and some conventional semen parameters or
CASA profiles. We also found some parameters of exposed
workers (sex chromosome disomy, nullisomies of sex chromosomes and chromosome 18) were significantly higher than
those of the external controls but not the internal controls. And
these data of internal cohorts were also higher than external
cohorts. These facts may be caused by light adverse chemical
exposure in the internal area, sample size limiting, ages of the
donors, working environment, lifestyle, economic status, and
other individual characteristics. Although sex chromosomes
are thought to be more susceptible to aneuploidy, due to the
presence of a single chiasma between these chromosomes at
meiosis I (Hassold et al., 1991), we could not rule out the
possibility that chromosome 18, considered in this study, may
be susceptible to carbaryl exposure because Egozcue et al.
(2000) reported that chromosome 21 displayed a higher incidence of disomy among autosomes in a sperm-FISH study.
Some confounding factors may contribute to the genotoxicity of carbaryl. Previous data showed that smoking and
alcohol consumption are associated with an increased risk of
aneuploidy and DNA fragmentation. Chemicals in cigarette are
also found to be able to reach to the male reproductive system,
increasing damage to sperm and lowering seminal quality
(Harkonen et al., 1999; Naccarati et al., 2003; Shi et al., 2001;
Sun et al., 1997). In this study, we only recruited the donors
who were nonsmokers and not regular drinkers. Age is also
a potential impact factor of the chromosomal aneuploidy,
especially sex chromosomes (Lowe et al., 2001; Naccarati
et al., 2003; Robbins et al., 1997), though other studies showed
no connections between age and aneuploidy (Harkonen et al.,
1999; Luetjens et al., 2002). In our study, the distribution of age
showed no evidence of differences among three groups.
We also found the age of donors had no significant associations with sperm aneuploidy. Our aneuploid results, however,
were higher than former reports (Padungtod et al, 1999; Shi
et al., 2001). The differences of methodology, observation,
nutritional habits, race, and region may contribute to these
results.
622
XIA ET AL.
Our study provides direct evidence that carbaryl has
potential genotoxic effects such as DNA fragmentation and
CA on human spermatozoa from exposed workers, and these
effects might be associated with adverse reproductive outcomes. These results might also apply to both the occupational
pesticide exposure and the environmental pesticide exposure
from crop-dusting or daily-life.
Grover, I. S., Ladhar, S. S., and Randhawa, S. K. (1989). Carbaryl-A selective
genotoxicant. Environ. Pollut. 58, 313–323.
ACKNOWLEDGMENTS
Harkonen, K., Viitanen, T., Larsen, S. B., Bonde, J. P., and Lahdetie, J. (1999).
Aneuploidy in sperm and exposure to fungicides and lifestyle factors.
Environ. Mol. Mutagen. 34, 39–46.
The authors wish to acknowledge Lifeng Tan and Wei Wu for their help
during this study. This project was supported by the Preliminary Study of an
Important Project in the National Basic Research (No.200150) and the
Greatest Project in the National Basic Research (No.2002CB512908).
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