Genotoxic Effects on Spermatozoa of Carbaryl-Exposed Workers
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《毒物学科学杂志》
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
ABSTRACT
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 = 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 = 0.035 and p = 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 = 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 = 0.564 and r = 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 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.
INTRODUCTION
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 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 x 10–3 mg/m3, which was significantly higher than those in the internal control area (6.30 x 10–3 mg/m3, p < 0.01) and external control area (undetected). Simultaneously, the mean concentrations expressed as a time-weighted 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 air-dried. 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, +18/100; 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.
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 Ca2+- and Mg2+-free phosphate-buffered saline (PBS) at 4°C. Sperm suspension with appropriate concentration (about 3 x 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 μl 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 μl 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 2x 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 2x SSC at 37°C for 30 min, sperm slides were denatured in a solution of 70% formamide/2x 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 μl of D18Z1 probe (chromosome 18 satellite probe, direct blue, Qbiogene, Montreal, Canada) and 5 μl 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 (0.5x SSC/0.1%SDS) at 65°C and 1x phosphate-buffered detergent (PBD, Qbiogene, Montreal, Canada) at RT for 5 min each. The slides were counterstained by 10 μl 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 four-fold band filter set with 1000x 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 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 group and control groups. However, the morphological defects as sperm abnormalities (including head abnormality, neck/mid-piece abnormality, tail abnormality, and mixed abnormality) in the carbaryl-exposed group were significantly higher than those in the external control group (p = 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).
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 = 0.035 and p = 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 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 carbaryl-exposed 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 carbaryl-exposed 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 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 = 0.564 and r = 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 = 0.008). There was also positive correlation between sex chromosome disomy, aneuploidy rate, and sperm abnormality (r = 0.564 and r = 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 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.
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 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 observations 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.
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.
NOTES
The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.
ACKNOWLEDGMENTS
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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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
ABSTRACT
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 = 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 = 0.035 and p = 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 = 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 = 0.564 and r = 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 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.
INTRODUCTION
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 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 x 10–3 mg/m3, which was significantly higher than those in the internal control area (6.30 x 10–3 mg/m3, p < 0.01) and external control area (undetected). Simultaneously, the mean concentrations expressed as a time-weighted 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 air-dried. 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, +18/100; 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.
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 Ca2+- and Mg2+-free phosphate-buffered saline (PBS) at 4°C. Sperm suspension with appropriate concentration (about 3 x 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 μl 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 μl 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 2x 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 2x SSC at 37°C for 30 min, sperm slides were denatured in a solution of 70% formamide/2x 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 μl of D18Z1 probe (chromosome 18 satellite probe, direct blue, Qbiogene, Montreal, Canada) and 5 μl 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 (0.5x SSC/0.1%SDS) at 65°C and 1x phosphate-buffered detergent (PBD, Qbiogene, Montreal, Canada) at RT for 5 min each. The slides were counterstained by 10 μl 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 four-fold band filter set with 1000x 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 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 group and control groups. However, the morphological defects as sperm abnormalities (including head abnormality, neck/mid-piece abnormality, tail abnormality, and mixed abnormality) in the carbaryl-exposed group were significantly higher than those in the external control group (p = 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).
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 = 0.035 and p = 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 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 carbaryl-exposed 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 carbaryl-exposed 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 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 = 0.564 and r = 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 = 0.008). There was also positive correlation between sex chromosome disomy, aneuploidy rate, and sperm abnormality (r = 0.564 and r = 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 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.
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 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 observations 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.
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.
NOTES
The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.
ACKNOWLEDGMENTS
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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