Article pubs.acs.org/est
Endosulfan Isomers and Sulfate Metabolite Induced Reproductive Toxicity in Caenorhabditis elegans Involves Genotoxic Response Genes Hua Du, Min Wang, Hui Dai, Wei Hong, Mudi Wang, Jingjing Wang, Nanyan Weng, Yaguang Nie, and An Xu* Key Laboratory of Ion Beam Bioengineering, Hefei Institutes of Physical Science, CAS and Anhui Province, Hefei, Anhui, PR China ABSTRACT: Endosulfan is enlisted as one of the persistent organic pollutants (POPs) and exists in the form of its α and β isomers in the environment as well as in the form of endosulfan sulfate, a toxic metabolite. General endosulfan toxicity has been investigated in various organisms, but the effect of the isomers and sulfate metabolites on reproductive function is unclear. This study was aimed at studying the reproductive dysfunction induced by endosulfan isomers and its sulfate metabolite in Caenorhabditis elegans (C. elegans). We also determined a role for the DNA-damage-checkpoint gene hus-1. Compared to β-endosulfan and its sulfate metabolite, α-endosulfan caused a dramatically higher level of germ cell apoptosis, which was regulated by DNA damage signal pathway. Both endosulfan isomers and the sulfate metabolite induced germ cell cycle arrest. Loss-of-function studies using hus-1, egl-1, and cep-1 mutants revealed that hus-1 specifically influenced the fecundity, hatchability, and sexual ratio after endosulfan exposure. Our data provide clear evidence that the DNA-checkpoint gene hus-1 has an essential role in endosulfan-induced reproductive dysfunction and that α-endosulfan exhibited the highest reproductive toxicity among the different forms of endosulfan.
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in a Bacillus stearothermophilus culture.11,12 In zebrafish, although more physical abnormalities were observed in α-endosulfanexposed larvae, endosulfan sulfate also disrupted embryonic development.13 A 48h LC50 assay of Oncorhynchus mykiss indicated that endosulfan sulfate appeared to be as toxic as β-endosulfan.11 As a highly lipophilic compound, endosulfan persists in the seminal vesicles and epididymis, inducing testicular impairment and sperm abnormalities with chronic exposure.14 Endosulfan exposure reduces egg hatching and alters germ cell distribution in Drosophila melanogaster and zebrafish, respectively, and also reduces the sperm motility rate in mice.15−17 An epidemiological study carried out to assess the potential effects of aerial spraying of endosulfan on male reproductive development suggested that endosulfan exposure may delay sexual maturity and interfere with sex hormone synthesis in male children.18 A survey in estuarine and near-coastal zones indicates that endosulfan sulfate constitutes over 80% of the organochlorine pesticides (OCP) in the gonads of Cynoscion guatucupa.19 Nevertheless, the effect of endosulfan and its byproduct on reproductive functions needs further elucidation. Considering that previous sublethal studies were based on long-term exposure experiments,15,20,21 there is an urgent requirement to evaluate endosulfan-induced reproductive
INTRODUCTION Endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9a-hexahydro6,9-methano-2,3,4-benzodioxyanthiepin-3-oxide) is a widely used organochlorine pesticide, which produces toxic metabolites that pose environmental hazards.1 Technical grade endosulfan is composed of two stereoisomers, α-endosulfan and β-endosulfan, that occur in ratios ranging from 2:1 to 7:3.2 Environmental degradation forms less toxic and hydrophilic isomers such as endosulfan diol, lactona, ether, and hydroxy ether, but biological transformation produces the more toxic metabolite, endosulfan sulfate.3,4 There is evidence indicating that technical grade endosulfan can transform to endosulfan sulfate in organisms after 24 h exposure.5 Because of its extensive use, bioaccumulation, and toxicity in the ecosystem, endosulfan and endosulfan sulfate are included on the Stockholm Convention list of persistent organic pollutants (POPs) and are being phased out globally.6 Despite the ban, endosulfan residues are abundant in different matrices including water, soil, fruit, milk, and human blood. Therefore, a deeper knowledge of the toxic mechanism of endosulfan isomers and the sulfate metabolite may provide information critical for assessment of the potential risk to environment and human life. The toxic effects of endosulfan have been investigated in target and nontarget organisms, which have revealed that endosulfan isomers and its sulfate metabolite exhibit different levels of toxicity.7−9 Endosulfan isomers and endosulfan sulfate cause genotoxicity in mammalian cells.10 However, compared to βendosulfan, the α isomer exhibited higher lethal effects on rainbow trout, Daphnia, and Hyalella and increased the lag phase © 2015 American Chemical Society
Received: Revised: Accepted: Published: 2460
October 3, 2014 January 15, 2015 January 22, 2015 January 22, 2015 DOI: 10.1021/es504837z Environ. Sci. Technol. 2015, 49, 2460−2468
Article
Environmental Science & Technology
appeared yellow-green after AO staining, representing increased DNA fragmentation, whereas intact cells were uniformly green. Mitotic Cell Nuclei Determination. The worms were fixed with Carnoy’s fixative (six parts ethanol, three parts chloroform, and one part glacial acid) and air-dried. The gonads were stained with 2 μg/mL DAPI. The nuclei in the mitotic zone of the germline were quantified under a fluorescence microscope. Fecundity and Hatchability Assays. Fecundity and hatchability were measured as previously described.25 Briefly, L1-stage worms were exposed to endosulfan for 60 h and then moved to a new plate every 24 h until they stopped laying eggs. All eggs were cultured at 20 °C for 24 h. The number of worms at all stages and the number of eggs were counted under a dissecting microscope. Offspring that hatched over 6 days were documented as the total fecundity. The ratio of the hatched worms to the total eggs laid (hatched and unhatched) was enumerated as the hatchability. Data Analysis. All values were expressed as mean ± standard error. Statistical differences (p < 0.05) between various concentrations of different strains were tested using analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. To compare the results for different strains, statistical analysis was carried out with a two-way ANOVA with Dunnett’s t test.
toxicity during a whole-developmental-period exposure in a relatively short time. Therefore, in this study, we examined the effects of endosulfan on reproductive function in Caenorhabditis elegans. As a nematode, it represents organisms that are ubiquitously present in the ecosystem.22,23 The transparent body allows viewing of cellular activities at organism level, and genetic knockout strains and reverse genetic tools for screening signaling pathway responses to environmental stresses are available. C. elegans also has a short life span, gives rise to a large number of offspring, and contains almost all the genes needed for apoptosis regulation in humans, which makes it an excellent tool to elucidate the mechanisms of the reproductive dysfunction caused by endosulfan isomers and the sulfate metabolite.24 We proved that α-endosulfan and β-endosulfan, and their sulfate metabolite, induced a significant increase of germ cell apoptosis and germ cell cycle arrest and decreased fecundity and hatchability. α-Endosulfan exhibited the highest reproductive toxicity. Moreover, the DNA-damage-checkpoint gene, hus-1, mediated these effects in a dose-dependent way. Interestingly, the percentage of males greatly increased in hus-1 mutants exposed to α-endosulfan.
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MATERIALS AND METHODS Chemicals. High-purity α-endosulfan, β-endosulfan, endosulfan sulfate, and 5-fluoro-2′-deoxyuridine (5-FudR) were purchased from Sigma-Aldrich (St. Louis, MO). Acridine orange (AO) and 4′,6′-diamidino-2-phenylindole (DAPI) were commercially obtained from Molecular Probes (Eugene, OR). Worm Strains and Culture. All worm strains were cultured at 20 °C in petri dishes on nematode growth medium (NGM) seeded with Escherichia coli (E. coli) OP50, according to standard protocols. The wild-type strain used was Bristol N2. The following strains were provided by the Caenorhabditis elegans Genetics Centre (CGC): N2, ced-3(n717), ced-4(n1162), ced9(n1950), cep-1(w40), egl-1(n487), hus-1(op244), hus-1(op241), and hus-1::GFP(opIs34). Worm Exposure. α-Endosulfan, β-endosulfan, and endosulfan sulfate were dissolved in dimethyl sulfoxide (DMSO). The stock solution was diluted in M9 Buffer (3 g of KH2PO4, 6 g of Na2HPO4, 5 g of NaCl, 1 mL of 1 M MgSO4) to 0.1, 1, and 10 μM working concentrations. Young adult hermaphrodites were transferred into Costar 24-well tissue plates with either M9 buffer or test solution for 12 h. Escherichia coli OP50 was added as a food source. The DMSO maximum concentration (0.01%) in the endosulfan work solution did not affect the worms. Life-Span Assay. All life-span assays were conducted in 96-well plates containing M9 buffer with 5-FudR. E. coli OP50 was added as a food source. Briefly, 48 h after hatching, 30 agesynchronized hermaphrodites were picked and transferred to a 96-well plate. Each well contained one worm in 200 μL M9 or test solution at 20 °C. 5-FudR (20 μg/mL) was added to each well to prevent offspring generation. All worms were continuously exposed to endosulfan until the worm was dead, which was identified by the lack of response to mechanical stimulation. Apoptosis Assay. Apoptotic germ cells were measured by acridine orange (AO) vital staining. Briefly, synchronized worms at the L4 stage of development were exposed to graded concentrations of endosulfan (0.1−10 μM) and stained with 500 μL of 25 μg/mL AO and OP50. After 60 min of incubation at 20 °C, the worms were transferred onto bacterial lawns for recovery. The worms were immobilized by levamisole, and fluorescent staining was observed under an Olympus 1 × 71 microscope (Olympus, Tokyo, Japan). The apoptotic cells
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RESULTS AND DISCUSSION Endosulfan Isomers and the Sulfate Metabolite Reduced Life Span. To examine the toxic effects of endosulfan on the life cycle, L3 stage worms were exposed to α-endosulfan, β-endosulfan, and endosulfan sulfate at 1 μM for their whole life. The average life span of N2 worms was 19 d in M9 buffer containing E. coli OP50 at 20 °C. However, exposure to α-endosulfan, β-endosulfan, and endosulfan sulfate decreased the average life span to 16.03, 17.23, and 18.43 d, respectively (Figure 1). This finding indicated that C. elegans is sensitive to the
Figure 1. Effect of exposure to 1 μM endosulfan isomers and the sulfate metabolite on the life span of wild type N2 worms. Worms were exposed beginning at the L3 stage until the end of their life. Thirty worms were scored for each group.
toxic effects of endosulfan isomers and the sulfate metabolite. Therefore, it can be reliably used to study the toxic response to endosulfan. In C. elegans, removing the germ cells extends the lifespan by triggering nuclear localization and activation of the insulinsignaling downstream target DAF-16/FOXO transcription factor in the intestine.26 Insulin-like growth-factor signaling 2461
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Figure 2. Average number of endosulfan-induced germline apoptotic cells in wild type N2 and mutant strains ced-9(n1950), ced-4(n1162), and ced3(n717). The strains were exposed to varying endosulfan concentrations (0.1, 1, and 10 μM). (A and B) α-Endosulfan-induced germline apoptosis in N2 worms at 0.0 and 10 μM, respectively; white arrowheads indicate the apoptotic cells. (C) Endosulfan-induced dose-dependent germ cell apoptosis in N2 strain. (D) α-Endosulfan, (E) β-endosulfan, and (F) endosulfan sulfate induced germline apoptotic cells in mutant strains ced-9(n1950). For comparison, the same data for N2 wild type, as shown in A, were presented in all figures. The data represent the average of three independent experiments. Error bars indicate ± SD; asterisks indicate statistical significance at p < 0.05.
the C. elegans, as revealed by AO vital staining (Figure 2A,B). Moreover, there was a dose-dependent increase in germ cell corpses in worms exposed to α-endosulfan and β-endosulfan (Figure 2C). Even at the lowest concentration (0.1 μM), a significant increase in apoptotic germ cell corpses was observed in C. elegans exposed to parent isomers. In contrast, endosulfan sulfate did not affect the induction of germ cell corpses at 0.1 μM. At higher concentrations (1 and 10 μM), both endosulfan
engages evolutionarily conserved mechanisms for the control of aging.27 Hence, we hypothesized that endosulfan would alter the fate of germ cells in C. elegans. Endosulfan Isomers and the Sulfate Metabolite Increased Germ Cell Apoptosis. A recent study showed that a subacute concentration of endosulfan can induce apoptosis in Sertoli and Leydig cells.28 Endosulfan exposure significantly increased germline apoptosis in the meiotic zone of the gonad of 2462
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Figure 3. Effect of 0.1 and 1 μM endosulfan on germline apoptosis in genotoxic-response-gene mutant hus-1, p53-like-gene mutant cep-1, and BH3-onlydomain mutant egl-1. (A) α-Endosulfan, (B) β-endosulfan, and (C) endosulfan sulfate induced apoptosis in genotoxic-response-gene mutant strains. (D) Number of HUS-1::GFP foci per 40 germ cells in control and endosulfan-exposed groups. (E) HUS-1::GFP is diffused in the control. (F) Relocalized HUS-1::GFP is seen as bright green foci. Images were acquired with Carl Zeiss LSM 710 laser-scanning confocal microscope using 63× (oil) magnification. Arrowheads indicate HUS-1::GFP foci. Data were pooled from three independent experiments. Error bars indicate ± SD; asterisks indicate statistical significance at p < 0.05.
ced-4(n1162), ced-3(n717), and gain-of-function mutants ced9(n1950) were exposed to endosulfan. Although few germline cell corpses were observed in the ced-3(n717) and ced-4(n1162) mutants, the ced-9(n1950) gain-of-function mutants showed a much higher spontaneous cell death than the wild type N2 worms. The germ cell corpses induced by α-endosulfan, β-endosulfan, or endosulfan sulfate were nearly abolished in ced-3(n717) and ced-4(n1162) mutants, indicating that CED-3 and CED-4 were essential for endosulfan-induced apoptosis. In contrast, the number germ cell corpses induced by endosulfan
isomers as well as the metabolite induced a dramatic increase of germ cell corpses, though α-endosulfan proved to have the most adverse effect. Caspase protein CED-3 and Apaf-1 homologue CED-4 are required for apoptosis in C. elegans, and CED-9 may prevent death by sequestering CED-4 and proCED-3 in an inactive ternary complex, the apoptosome. During germline apoptosis, CED-3 is activated by CED-4, which is constitutively inhibited by CED-9.29,30 To investigate the core apoptosis signaling pathway in endosulfan-exposed germ cells, loss-of-function mutants 2463
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transition nuclei.33 Irradiation-induced DNA damage can arrest the germline cell cycle in C. elegans.41 The above evidence links DNA damage to germline apoptosis, therefore, we anticipated the induction of germ cell cycle arrest by endosulfan. In fact, after the young adult hermaphrodites were exposed to different concentrations of endosulfan for 24 h, the numbers of mitotic nuclei per gonad arm decreased in all groups (Figure 4C). At all tested concentrations, α-endosulfan led to the
was only slightly altered in ced-9(n1950), indicating that Bcl-2like protein CED-9 acted as an antiapoptotic regulator in endosulfan-induced apoptosis (Figure 2D,E,F). These data suggested that endosulfan isomers and the metabolite elicited programmed germ cell death in C. elegans in a dose-dependent way; this was consistent with observations in Jurkat T-cells and Nile tilapia (Oreochromis niloticus) splenocytes.31,32 DNA damage or nongenotoxic factors as well as environmental stresses such as radiation, heavy metals, and chemicals, can also induce germline apoptosis.33−35 Germline apoptosis has an integral role in human oogenesis. The evolutionary conservation of the key regulators between humans and C. elegans makes the C. elegans germline a valuable system for understanding the mechanisms involved in endosulfan-induced toxicity of the reproductive system in humans. Regulation of Genotoxic-Response Genes in EndosulfanInduced Germline Apoptosis. Endosulfan can cause sisterchromatid exchanges, micronuclei, and DNA strand breaks.36 The C. elegans gene egl-1, encoding the BH3-domain protein, is required for apoptosis under genotoxic stress, whereas p53, CEP-1, induces germ cell death by activating the transcription of the target genes egl-1 and ced-13.37,38 In C. elegans, hus-1 is the checkpoint gene that is required for DNA-damage-induced apoptosis.39 Germline apoptosis was not altered in the loss-offunction mutants egl-1(n487), hus-1(op241), and cep-1(w40) exposed to α-endosulfan as compared to the control, revealing that these DNA-damage-response genes were indispensable for α-endosulfan-induced apoptosis (Figure 3A). However, the egl-1(n487), hus-1(op241), and cep-1(w40) mutants exposed to β-endosulfan and endosulfan sulfate at 1 μM showed a significant increase in the apoptotic germ cells (Figure 3B,C). In C. elegans, HUS-1::GFP is diffused in proliferating germ nuclei under normal conditions. However, it localizes in the nucleus in response to DNA damage, concentrating at distinct nuclear foci that are considered to be DNA breakage points. Thus, the nuclear localization of HUS-1 implies DNA damage, and the foci likely represent sites of double-strand breaks (DSBs).39 The opIs34[hus-1::gf p] strain was used to confirm endosulfan-induced DNA damage. Compared to the few occasional HUS-1::GFP foci in the pachytene cells of the germline, endosulfan treatment caused a dramatic increase in HUS-1::GFP foci in the germline (Figure 3E,F). The ratio of cells containing spontaneous HUS-1::GFP foci was 0.18 ± 0.03. As shown in Figure 3D, the ratios of cells containing HUS1::GFP foci increased significantly to 4.3 ± 0.53 and 4.12 ± 0.4 with 0.1 and 1 μM α-endosulfan, respectively. Although βendosulfan and endosulfan sulfate resulted in a much lower number of HUS-1::GFP positive cells than α-endosulfan, a significant increase of HUS-1::GFP foci was clearly observed. These results indicated that both endosulfan isomers and the sulfate metabolite induce DNA damage in the reproductive system of worms. It has been reported that endosulfan exposure can induce DNA damage in D. melanogaster larvae.15 Herein, we provided a clear evidence that genotoxic response genes are critical in regulating endosulfan-induced germ-cell apoptosis, implying a close relationship between DNA damage and endosulfaninduced reproductive errors. Endosulfan Induced Germline Cell Cycle Arrest. Besides germ cell apoptosis, DNA damage checkpoints can induce cell cycle arrest in the worm germline.39,40 In the gonad of C. elegans hermaphrodites, mitotic nuclei are located at the distal end of the gonad and are distinguished by DAPI stain as crescent-shaped
Figure 4. Average number of mitotic cells after exposure to varying concentrations (0.1 and 1 μM) of endosulfan isomers and the sulfate metabolite. (A) Germline of C. elegans after DAPI staining in the control group. Image was taken by an Olympus 1 × 71 microscope. (B) Germline in C. elegans after exposure to 10 μM α-endosulfan for 24 h. Arrowheads indicate the transition zone. (C) Average number of mitotic cells in germline. Data were pooled from three independent experiments. Error bars indicate ± SD; asterisks indicate statistical significance at p < 0.05.
most significant decrease in the number of mitotic nuclei, followed by the β-endosulfan-exposed groups. Compared to the results obtained with α-endosulfan and β-endosulfan, 0.1 μM endosulfan sulfate did not induce significant changes in the number of mitotic nuclei. These findings indicated that exposure to endosulfan isomers and the metabolite could induce germline mitotic cell cycle arrest. Combined with the data from the apoptosis assay, it is reasonable to confirm that endosulfan-induced DNA damage might be responsible for the reproductive dysfunction in C. elegans. Effect of Genotoxic-Response Genes on Fecundity. Endosulfan exposure reduces germ cells in the reproductive system of male mice and increases abnormal sperm heads.42 To understand the effects of endosulfan exposure on the reproductive system in C. elegans, we tested worm fecundity after exposure, from the L1 larval stage to the adult stage. Exposure to all the compounds decreased the progeny of N2 worms in a dosedependent fashion. The total eggs laid during the 6 days decreased from 210.67 ± 5.51 in the control group to 147.67 ± 7.52 and 86 ± 1.41 at 0.1 and 1 μM α-endosulfan, respectively. The adverse effect on fecundity caused by β-endosulfan was less 2464
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Figure 5. Effect of endosulfan isomers and the sulfate metabolite on total fecundity of worms. (A) Total fecundity in wild type N2 strain. Total fecundity in genotoxic-response-gene mutant strains (B) hus-1(op241), (C) egl-1(n487), and (D) cep-1(w40). Data were pooled from three independent experiments. Error bars indicate ± SD; asterisks indicate statistical difference at p < 0.05.
significant than that of α-endosulfan. Although there was slight reduction of fecundity with 0.1 μM endosulfan sulfate, the decrease was conspicuous with 1 μM (Figure 5A). Because DNA damage is involved in endosulfan-induced apoptosis in germline cells, the worms carrying mutated alleles of egl-1(n487), hus-1(op241), and cep-1(w40) were exposed to endosulfan. Using hus-1(op241) mutants, we found that all endosulfan-exposed groups exhibited higher fecundity than the N2 strain, which was consistent with the observation in irradiated hus-1(op241) mutants.39 In addition, the extent that the fecundity decreased in hus-1(op241) mutants treated with endosulfan isomers and metabolite was lower than those in wild type N2 worms (Figure 5B). However, the endosulfan exposure resulted in similar effects on fecundity in egl-1(n487) and cep-1(w40) mutants and the N2 strain (Figure 5C,D). These findings revealed that endosulfan exposure reduced the fecundity of C. elegans, which is concurrent with the effects in worms exposed to other pesticides such as monocrotophos and chlorpyrifos.43,44 α-Endosulfan exhibited the most dramatic effect on fecundity. Fecundity of hus-1(op241) mutants exposed to endosulfan isomers and the metabolite was higher than that in exposed N2 strain. However, the compounds did not exert such an effect on the other two genotoxic-response-gene mutants (egl-1 and cep-1), which might be due to the differential effects on different genes. It is possible that loss of hus-1 function damaged
germ cells that escaped from apoptosis, resulting in more germ cells differentiating to eggs. Thus, the data indicate that the DNA-damage-checkpoint gene hus-1 was specifically involved in endosulfan-induced fecundity reduction. Effect of Genotoxic-Response Genes on Hatchability. In comparison to the decreased fecundity, the ratio of abnormal offspring increased more dramatically as a consequence of endosulfan exposure.45 In zebrafish, abnormal responses including paralysis and pericardial and yolk sac edema are the most sensitive end point following α-endosulfan and endosulfan sulfate exposure.13 To evaluate the effects of endosulfan on the prior-hatch development, hatchability was measured in C. elegans. Egg hatchability of the wild type N2 worms decreased with 1 μM endosulfan exposure, particularly in the initial 3 days (Figure 6A). The reduction in hatchability was more significant in hus1(op241) worms that did not recover compared to the wild type N2 worms. Although the hatchability of the egl-1(n487) and cep-1(w40) mutants after endosulfan exposure was slightly lower than that of the wild-type strain, it was restored to the control level by the fourth day (Figure 6C,D). Endosulfan also reduces the emergence of D. melanogaster and decreases the hatchability of Palaemonetes pugio.15,46 We observed a dose-dependent reduction in hatchability after exposure to endosulfan isomers and the metabolite. Because hus-1 mutants have abnormal levels of embryonic lethality, this strain is more sensitive to the 2465
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Figure 6. Effect of 1 μM endosulfan isomers and the sulfate metabolite on daily hatchability of worms. (A) Daily hatchability of wild type N2 worms. Daily hatchability of mutants (B) hus-1(op241), (C) egl-1(n487), and (D) cep-1(w40). Data were pooled from three independent experiments. Error bars indicate ± SD.
embryonic-lethal effects of ionizing-radiation-induced DNA damage.39 Accordingly, we showed that the endosulfan-induced reduction in the hatchability of the hus-1(op241) strain was significantly higher than that of the wild type N2 strain. However, the hatchability of egl-1 and cep-1 mutants and the N2 worms exhibited comparable endosulfan sensitivity. Thus, our results implied that among the tested genotoxic response genes, checkpoint gene hus-1 has a unique role in endosulfan-induced hatchability decrease. Furthermore, α-endosulfan caused more severe impairments in the offspring compared to the β-endosulfan and endosulfan sulfate effects. Effects of Endosulfan Isomers and the Metabolite on the Sex Ratio. C. elegans exist as either self-fertile hermaphrodites or males. The primary sex-determination signal is the ratio of X chromosomes to autosomes. Hermaphrodites normally have two X chromosomes (XX) and males have one (XO).47 In N2 worms, male progeny is less than 0.2%. Barring a slight increase in male progeny in worms exposed to 1 μM α-endosulfan, other concentrations of endosulfan did not affect the proportion of males in N2 worms (Table 1). However, the hus-1(op244) mutant sex ratio significantly changed within all endosulfan-exposed groups. The proportion of males in the 1 μM α-endosulfan-exposed group (5.2% ± 1.2%) was 4.3-fold higher than that in the control (1.2% ± 0.3%). At the same concentrations, β-endosulfan and endosulfan sulfate increased male proportion by 2.1-fold and 1.5-fold, respectively. However, endosulfan exposure resulted in similar effects on sex ratio in
Table 1. Percent Males in N2 and Genotoxic Response Gene Mutants after Exposure to 0.1 and 1 μM Endosulfan Isomers and Sulfate Metabolite concentrations (μM) strains N2
hus-1(op244)
egl-1(n487)
cep-1(w40)
chemicals
control
0.1
1
α-endosulfan β-endosulfan endosulfan sulfate α-endosulfan β-endosulfan endosulfan sulfate α-endosulfan β-endosulfan endosulfan sulfate α-endosulfan β-endosulfan endosulfan sulfate
0.15 ± 0.0 0.15 ± 0.0 0.15 ± 0.0 1.2 ± 0.3 1.3 ± 0.3 1.2 ± 0.2 0.25 ± 0.0 0.25 ± 0.0 0.25 ± 0.0 0.2 ± 0.0 0.25 ± 0.0 0.2 ± 0.0
0.15 ± 0.0 0.15 ± 0.0 0.15 ± 0.0 3.4 ± 0.7 1.8 ± 0.3 1.4 ± 0.4 0.33 ± 0.1 0.3 ± 0.1 0.25 ± 0.0 0.3 ± 0.1 0.3 ± 0.1 0.25 ± 0.0
0.20 ± 0.0 0.15 ± 0.0 0.15 ± 0.0 5.2 ± 1.2 2.7 ± 0.7 1.8 ± 0.5 0.5 ± 0.15 0.35 ± 0.1 0.3 ± 0.0 0.35 ± 0.1 0.33 ± 0.1 0.3 ± 0.1
egl-1(n487) and cep-1(w40) mutants and wild type N2 strain (Table 1). The hus-1(op244) mutants have high levels of chromosomal nondisjunction, which leads to high proportion of males and sensitivity to chromosomal aberrations.39 In other organisms such as Daphnia magna, exposure to endosulfan sulfate can increase the production of males.45 This study showed that compared to wild type N2 worms, the hus-1(op244) loss-of-function mutants had 2466
DOI: 10.1021/es504837z Environ. Sci. Technol. 2015, 49, 2460−2468
Article
Environmental Science & Technology
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a significant increase in males after endosulfan exposure, which was different from the other two genotoxic-response-gene mutants (cep-1 and egl-1). This finding implied that endosulfan not only caused DNA damage but also affected meiotic chromosomal disjunction. Moreover, hus-1 in particular is involved in resisting endosulfan-induced chromosomal aberration. Endosulfan is a POP that is ubiquitously found in various geographical regions.48,49 Of the various detrimental effects, reproductive toxicity is a fundamental response to endosulfan exposure.28,35 Moreover, DNA damage has been suspected to be one of the causative mechanisms linked to endosulfan toxicity.10,50 In this study, we demonstrated that C. elegans is a suitable in vivo model to investigate reproductive toxicities induced by endosulfan isomers and the metabolite. Both endosulfan isomers and the metabolite lead to reproductive system dysfunction, which was mediated by the DNA-damagecheckpoint gene hus-1. However, the internal relation between conformation of endosulfan and the genotoxicity requires further investigation.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +86 (551) 65593336. Fax: +86 (551) 65595670. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the Caenorhabditis elegans Genetics Center, USA, for providing most of the C. elegans strains. We thank Prof. Ge Shan and Dr. Tom K. Hei for suggestions. The work herein was partially supported by grants from Major National Scientific Research Projects, 2014CB932002; the Strategic Leading Science & Technology Program (B), XDB14030502; the National Natural Science Foundation of China, U1232144 and 30570442; and the Major/Innovative Program of Development Foundation of Hefei Center for Physical Science and Technology, 2014FXCX010.
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