Induction of Germline Cell Cycle Arrest and Apoptosis by Sodium

mitotic cell cycle arrest and germline apoptosis, which, to some extent, exhibited a concentration- ... induce gene amplification, arrest cells in mit...
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Chem. Res. Toxicol. 2007, 20, 181-186

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Induction of Germline Cell Cycle Arrest and Apoptosis by Sodium Arsenite in Caenorhabditis elegans Shunchang Wang,†,‡,§ Ye Zhao,†,§ Lijun Wu,*,† Mingli Tang,† Caixing Su,† Tom K. Hei,| and Zengliang Yu† Key Laboratory of Ion Beam Bioengineering, Institute of Plasma Physics, Chinese Academy of Sciences, Hefei, Anhui 230031, People’s Republic of China, Department of Chemistry and Biology, Huainan Normal UniVersity, Huainan, Anhui 232001, People’s Republic of China, and Center for Radiological Research, College of Physicians and Surgeons, Columbia UniVersity, New York 10032 ReceiVed August 18, 2006

The nematode Caenorhabditis elegans has been shown to be a model organism in studying aquatic toxicity. Although epidemiological studies have shown that arsenic is teratogenic and carcinogenic to humans, the lethality assay indicated that C. elegans is less sensitive to inorganic arsenic than any other organisms that have been tested thus far. In the present study, we used the more malleable germline of C. elegans as an in vivo system to investigate the genotoxic effects of arsenite. After animals were exposed to sodium arsenite at concentrations ranging from 1 µM to 0.5 mM, mitotic germ cells and germline apoptosis were scored after DAPI staining and acridine orange vital staining, respectively. DMSO rescue experiments were performed by exposing C. elegans to 0.01 mM arsenite in the presence of DMSO (0.1%) for 24 h, and reactive oxygen species (ROS) were semiquantified by CM-H2DCFDA vital staining. The results indicated that arsenic exposure reduced the brood size of C. elegans and caused mitotic cell cycle arrest and germline apoptosis, which, to some extent, exhibited a concentration- and time-dependent manner. The addition of 0.1% DMSO completely rescued arsenic-induced cell cycle arrest and partially suppressed germline apoptosis. Furthermore, treatment of animals with arsenite at a dose of 0.01 mM significantly increased ROS production in the intestine, which could be reduced by DMSO treatment. The present study also indicated that C. elegans might be used as an in vivo model system to study the mechanisms of arsenic-induced genotoxic effects. Introduction Inorganic arsenic is ubiquitous in the environment either as compounds of arsenite (As3+) or arsenate (As5+). Epidemiologic data have shown that chronic exposure to arsenic in drinking water represents a significant health problem for people around the world and is associated with liver injury, peripheral neuropathy, and an increased incidence of cancer of the lung, skin, bladder, and liver (1, 2). In China, water born arsenism has been identified in at least eight provinces, affecting over 2 300 000 people, among which over 520 000 people are exposed to arsenic levels exceeding 50 µg/L (3). In certain parts of Bangladesh and West Bengal, India, as many as 5% of sampled drinking wells have arsenic levels exceeding 1 mg/L, and 27% of wells have levels exceeding 300 µg/L (4). To have a better treatment and prevention program for arsenic-associated diseases, there have been many studies conducted to provide information on the carcinogenic/genotoxic effects of arsenic in vitro. It was shown that exposure to arsenic induced apoptosis in certain tumor cell lines as well as normal cells (5, 6). Arsenic and arsenical compounds induce morphological transformation in murine C3H 10T1/2 cells and mutations in the CD59 gene locus in the highly sensitive AL cell system (7, 8). Arsenical compounds have also been shown to * To whom correspondence should be addressed. Tel: 86-551-5591602. Fax: 86-551-5591310. E-mail: [email protected]. † Chinese Academy of Sciences. ‡ Huainan Normal University. § These authors contributed equally to this paper. | Columbia University.

induce gene amplification, arrest cells in mitosis, inhibit DNA repair, and induce expression of the c-fos gene and the oxidative stress response genes in mammalian cells (9-11). Moreover, there is evidence that reactive oxygen species (ROS) mediate the biological effects of arsenic and that mitochondrial damage plays a crucial role in arsenic mutagenicity (12-14). These results strongly imply that arsenic is an important environmental hazard and ROS are involved in arsenite genotoxicity in the in vitro systems (8, 13). Moreover, using mouse system, Liu et al. (14) found that treatment of fertilized eggs of outbreed mice with 2-8 µg/mL arsenite reduced zygotes cleavage rate, increased apoptosis in blastocytes, and promoted telomere attrition. Navarro et al. (15) reported that arsernite disrupted miosis in CD-1 mouse oocytes and induced zygotes cell death at the dose of 8-16 mg/kg of body weight. However, as arsenical compounds always display pleiotropic effects in many experimental systems in which low levels (2 µM) of arsenite enhanced cell proliferation while high levels (40 µM) of arsenite induced cell apoptosis (16, 17), data about the mechanisms of carcinogenic/mutagenic effects of arsenic using in vivo animal models are still insufficient and the misconception that arsenic is a nongenotoxic carcinogen is still widely held. Thus, the employment of in vivo animal models to better understand the health impacts of arsenic exposure to both human and wildlife is much needed and indispensable. The nematode Caenorhabditis elegans has been widely used in developmental biology and genetics studies because of its ease of use, short lifespan, cellular simplicity, and genetic manipulability (18, 19). Studies in the past decades have

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demonstrated that C. elegans is a good model for environmental toxic bioassay (20, 21), stress response and aging (22, 23), drug screens (24), and genotoxic stress studies (25, 26). In the present study, to analyze the genotoxic effects of acute low dose arsenite exposure, we focused our attention on the C. elegans germline, which proliferates both during larval development and in adult worms. Also, in contrast to the almost invariant somatic C. elegans development, germline development is much more malleable (27) and is subjected to stochastic events, such as the elimination of many cells by programmed cell death to maintain tissue homeostasis (28).

Experimental Procedures Worms and Reagents. Wild-type N2 strain was provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources. Worms were cultured at 20 °C in Petri dishes on nematode growth medium (NGM) seeded with Escherichia coli strain OP50 as food (19). To obtain synchronized cultures, gravid hermaphrodites were lysed in an alkaline hypochlorite solution as described previously (29). Trivalent sodium arsenite (NaAsO2, AsIII) was a commercial product of Sigma Chemical (St. Louis, MO), and 4′,6′-diamidino-2-phenylindole (DAPI) and 5′,6′-chloromethyl-2′,7′dichlorodihydro-fluorescein diacetate (CM-H2DCFDA) were purchased from Molecular Probes (Eugene, OR). Worm Treatment. The procedures for animal handling and chemical exposure were conducted as described previously (30). Briefly, arsenic salts were dissolved in distilled water and diluted to final concentrations of 0.0, 0.001, 0.01, 0.1, and 0.5 mM in M9 buffer containing E. coli strain OP50 as a food source. Twenty synchronized young adult hermaphrodites were picked and transferred into a Costar 24 well tissue plates containing M9 buffer with or without test solutions. Worms were grown at 20 °C and removed at 6, 12, 24, and 36 h after arsenic exposure for further analysis. Brood Size Determination. A 1.0 mL aliquot of M9 buffer with or without test solutions was placed into a Costar 24 well plate as described by Thomas and Lockery (31). Single synchronized late young adult hermaphrodites were picked from NGM and transferred individually to each well. The cultures were maintained at 20 °C in an incubator and transferred daily to new wells until no new eggs were laid. The number of progeny was counted under a dissecting microscope 24 h after hatching. Mitotic Cell Nuclei Determination. For mitotic cell nuclei determination, 10 worms were picked out from test wells at indicated time points and suspended in 1 µL of distilled water, fixed with Carnoy’s fixative (six parts ethanol, three parts chloroform, and one part glacial acid), air-dried, and stained with a small drop of 2 µg/mL DAPI in M9 buffer as described by Gartner et al. (25). The nuclei in mitotic zone of the germline were counted under an Olympus 1 × 71 fluorescence microscope. Apoptosis Assay. Apoptotic germ cells were measured by acridine orange (AO, Sigma) staining using a procedure developed by Kelly et al. (32). Briefly, worms at indicated time points were picked from test wells into 1.5 mL brown tubes containing 100 µL of 25 µg/mL AO in M9 buffer and incubated for 2 h at 20 °C. The addition of bacteria in the buffer solution could facilitate uptake of the dye. Animals were allowed to recover for 10 min on bacterial lawns and then mounted in 60 µg/mL levamisole in M9 onto agar pads on microscope slides and examined by epifluorescence microscopy. The apoptotic cells appeared yellow or yellow-orange, representing increased DNA fragmentation, while intact cells were uniformly green in color (33). DMSO Treatment. To examine the possible role of ROS in arsenic-induced germline cell cycle arrest and apoptosis, 20 synchronized late L4 hermaphrodites were transferred to 0.01 mM arsenite containing buffer with or without 0.1% DMSO, a free radical scavenger. After 24 h of treatment, worms were picked out to examine the number of mitotic nuclei and apoptotic cells as described above.

Figure 1. Brood size of C. elegans exposed to different doses of arsenite. Single synchronized young adult hermaphrodites were transferred to a Costar 24 well microtiter plate at indicated concentrations of arsenite and transferred daily to new wells until no new eggs were laid. Data were expressed as percent of control. All values are represented by means ( SE; n ) 15. Error bars with different letters represent statistical significance (p value < 0.05).

ROS Determination. To quantify whether arsenic treatment increases ROS levels in C. elegans, animals were transferred to 1 mL of M9 buffer containing 1 µM CM-H2DCFDA and preincubated for 2.5 h at 20 °C. A 0.01 mM dose of arsenite with or without DMSO was then added and incubated for 30 min. Animals were mounted on 2% agar pads in 60 µg/mL levamisole and examined with a laser scanning confocal microscope (Leica, TCS SP2, Bensheim, Germany) at 488 nm of excitation wavelength and 510 nm of emission filter (14). The relative fluorescence intensities of ca. 120 µm body length near the anus were semiquantified using the Adobe Photoshop software. Data Analysis. All values were expressed as means ( standard error of the means. Significant differences at the P < 0.05 level were tested using ANOVA followed by a Dunnett t tests or twotailed Student’s t tests.

Results Arsenic Exposure Reduces the Brood Size of C. elegans. To determine whether arsenic exposure affected the fertility of C. elegans, each group of 15 young adult hermaphrodites was exposed individually to graded doses of trivalent sodium arsenite. All of the adult hermaphrodites survived during the experiments. As shown in Figure 1, exposing C. elegans to graded doses of arsenite ranging from 0.001 to 0.01 mM caused no significant changes in the brood size (279.5 ( 4.8 for control, 276.6 ( 9.3, and 267.2 ( 8.2 at 0.001 and 0.01 mM, respectively; p > 0.05), indicating that the fertility was not sensitive to low levels of arsenite. However, at doses of arsenite above 0.1 mM, the brood size of C. elegans decreased significantly as compared with the control (255.1 ( 8.9 at 0.1 and 250.3 ( 10.1 at 0.5 mM; p < 0.05). Arsenic Exposure Induces Germline Cell Cycle Arrest. In the gonad of C. elegans hermaphrodites, mitotic nuclei located at the distal end of the gonad and can be easily distinguished from the crescent-shaped transition nuclei after DAPI staining. To determine if arsenic exposure inhibited C. elegans mitotic cell division, young adult hermaphrodites were exposed to different concentrations of arsenite solutions for 24 h. The effects of graded doses of arsenite on mitotic cell division in C. elegans are shown in Figure 2A. The number of mitotic nuclei per gonad arm decreased significantly as compared to control (p < 0.05), suggesting that arsenite exposure could induce germline mitotic cell cycle arrest. The time exposure studies indicated that the mitotic cell cycle arrest could be detected as early as 12 h after 0.01 mM arsenite treatment, and the number of mitotic cells

Effects of Arsenite on C. elegans Germline

Figure 2. Arsenite-induced germline mitotic cell cycle arrest. (A) Synchronized young adult hermaphrodites were treated in M9 buffer with the indicated concentrations of arsenite for 24 h. Mitotic nuclei were stained with 2 µg/mL DAPI and viewed under a fluorescence microscope. (B) Worms were exposed to 0.01 mM sodium arsenite, and mitotic cells were scored at indicated time points. All values are represented by means ( SE; n ) 10. Error bars with different letters represent statistical significance (p value < 0.05).

per gonad arm exhibited a steady state of decrease thereafter and were significantly lower than that of their counterpart controls. The untreated control also exhibited a decrease in mitotic cells at 6 h but stopped decrease with time lapse (Figure 2B). Arsenic-Induced Germline Cell Apoptosis. To determine whether arsenic exposure induced apoptosis in the germline of C. elegans, adult young hermaphrodites were exposed to arsenic solutions as described above. The AO stained nuclei per gonad arm were counted after 24 h of exposure. As shown in Figure 3A, 0.001 mM arsenite exposure produced significant increase in apoptotic cells (2.2 ( 0.29 at 0 mM and 3.2 ( 0.32 at 0.001 mM; p < 0.05). These arsenite-induced germline apoptoses resulted in a dose-dependent increase when the arsenite concentration increased from 0.01 to 0.5 mM. To investigate the time course of arsenic-induced apoptosis, worms were treated with 0.01 mM arsenite and the apoptosis index was scored at different time points of 0, 6, 12, 24, and 36 h, respectively. The arsenite inducible germ cell apoptosis resulted in a dosedependent increase as well. Although the control group also showed a time-dependent increase due to the physiological germline cell apoptosis; nevertheless, the incidence was much lower than that of test groups (Figure 3B). DMSO Treatment Rescues Germline Cell Cycle and Apoptosis. Treatment of C. elegans with 0.1% DMSO alone for 24 h caused no genotoxic effects in terms of mitotic cells cycle arrest or germ cell apoptosis (Figure 4A,B). Arsenic treatment (0.01 mM for 24 h) induced a reduction in the number of mitotic nuclei per gonad arm to 86.2 ( 3.81. In the presence of 0.1% DMSO, the mitotic cells per gonad arm were restored

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Figure 3. Arsenite-induced germline cell apoptosis. (A) Synchronized young adult hermaphrodites were treated in M9 buffer with the indicated concentrations of arsenite for 24 h. Apoptotic cells were scored after staining with AO; only one gonad arm can be scored because the position of the autofluorescence intestine obscured one of the two gonad arms. (B) Worms were exposed to 0.01 mM sodium arsenite, and apoptotic cells were scored at indicated time points. All values are represented by means ( SE; n ) 10. Error bars with different letters represent statistical significance (p value < 0.05).

to control levels of 97.3 ( 2.41 (Figure 4A), indicating that the addition of 0.1%DMSO completely rescued arsenic-induced mitotic cell cycle arrest. Likewise, DMSO treatment restored the germ cell apoptosis level induced by arsenic to essentially control levels (4.9 ( 0.35 apoptotic cells as compared with 3.0 ( 0.26 when coexposed with 0.1%DMSO (p < 0.05, Figure 4B). Arsenite Exposure Increases the Generation of ROS. Figure 5 shows the fluorescent images of C. elegans prestained for 2.5 h with a 1 µM dose of the fluorescence probe, CM-H2DCFDA, and then exposed to a 0.01 mM dose of arsenite for 30 min. The strongest fluorescent signals were observed in the intestine, while weak fluorescence signals exhibited in oocytes, embryo, and germline (Figure 5C). The semiquantified ROS were expressed as arbitrary relative fluorescent units (RFU; Figure 6). In comparison with control, the RFU in the intestine increased 1.2-fold after treating the animals with arsenite at a dose of 0.01 mM. The arsenite-induced RFU decreased significantly when C. elegans were coexposed to DMSO (0.1%), indicating that the addition of DMSO significantly reduced the free radical induction by arsenite treatment.

Discussion Although arsenic is a well-established human carcinogen and has been shown to be genotoxic by a variety of in vitro studies (8, 11, 14), the mechanisms of its carcinogenicity remain unclear. Moreover, the paucity of available data based on in vivo systems makes it still difficult to assess the health risks of

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Figure 6. ROS production after exposure to arsenite. Relative fluorescent intensities in the C. elegans after exposing them to 0.01 mM arsenite either with or without 0.1% DMSO for 30 min. The relative fluorescence intensities are expressed in arbitrary units. All values are represented by means ( SE; n ) 10. An asterisk represents statistical significance (p value < 0.05) as indicated.

Figure 4. DMSO treatment rescued arsenic-induced mitotic cell cycle arrest and germline apoptosios. (A) Arsenic-induced mitotic cell cycle arrest was rescued by coexposure with 0.1% DMSO. (B) Coexposure with 0.1% suppressed arsenic induced germline apoptosis. All values are represented by means ( SE; n ) 10. An asterisk represents statistical significance (p < 0.05) as indicated.

Figure 5. Representative images of ROS production. C. elegans were pretreated with the fluorescent probe CM-H2DCFDA for 2.5 h and subsequently exposed to 0.1 mM arsenite for 30 min. (A) Control, (B) control at the presence of 0.1% DMSO, (C) 0.01 mM arsenite only, and (D) 0.01 mM arsenite with 0.1% DMSO.

arsenic to human as well as animals. C. elegans has been proven to be an excellent model to study the mechanisms of environmental stress response (34, 35). Because Williams and Dusenbery (36) proposed that C. elegans could be used as a model organism for aquatic toxicity tests, many researchers have

investigated its potential in various settings (23, 34, 35). Unfortunately, a 96 h LC50 test showed that C. elegans was less sensitive to inorganic arsenic than any other organisms that have been tested (30). The reported 24 h LC50 of C. elegans was 1.282 ( 0.306 mM for arsenite and 34.54 ( 3.261 mM for arsenate, respectively (37). In a recent 32 h toxicity assay based on solid agar medium, over 80% of the C. elegans survived at a test dose of 5 mM arsenite and nearly 100% survived at 3 mM (35). The fact that all adult worms survived at concentrations as high as 0.5 mM arsenite in this study suggests that lethality assay per se cannot reflect the genotoxic effects of arsenic on C. elegans. Therefore, it is desirable to employ more subtle end points to ascertain the DNA-damaging effects of arsenic in C. elegans. In contrast to the lack of lethality of arsenic treatment in adult worms, a nonlethal dose of arsenic reduced the brood size of C. elegans (Figure 1) and thus reduced the fertility, suggesting the potential genotoxic effects of arsenic to the germline of C. elegans. As an ubiquitous carcinogen and teratogen in the environment, arsenic has been shown to induce oxidative DNA damage and apoptosis at the dose of 0.1-1 µM (6, 38). Previous studies have shown that the germline of C. elegans, upon exposure to ionizing irradiation, undergo cell cycle arrest and apoptosis, which are spatially separated, resulting in a decreased number of mitotic germ cells and the removal of the cells that fail to be repaired properly (25). These findings suggest that it might be possible to use C. elegans as an in vivo model to study the mechanisms of the genotoxic effects of arsenite. The adult germline of C. elegans possesses a mitotic region at its distal end, which serves as stem cells that both self-renew and produce differentiating gametes. In young adults, the mitotic region is composed of over 100 germ cells per gonad arm, which are malleable under different environmental conditions (25, 39). By exposing C. elegans to different doses of arsenic and for various treatment periods, our data demonstrated that arsenic induced both mitotic cell cycle arrest and apoptosis in the germline. It is noteworthy that the cell cycle arrest effect of arsenite is independent of dose but remains robust at low levels (0.001 mM) and could be detected as early as 12 h after 0.01 mM arsenite treatment (Figure 2A,B). Furthermore, arsenite-induced germline apoptosis exhibits itself in a concentration-dependent, and to a certain extent, time-dependent manner. AO positively stained nuclei per gonad arm increased 1.8-fold when treated with 0.01 mM arsenite for 24 h (Figure 3). As compared with untreated control, arsenite treatment increased apoptotic cells

Effects of Arsenite on C. elegans Germline

at all treatment periods examined over their counterpart controls (Figure 3B). This finding indicated and confirmed previous observations made in mammalian cell lines (6, 38, 40) that arsenic exposure could induce germline apoptosis in C. elegans. Because the germ cells in the mitotic region serve as stem cells to produce differentiating gametes, the number of germ cells within the mitotic region normally keeps relatively constant without environmental disturbance (39). Thus, it should be notified that a reduction of mitotic cells observed in untreated controls from 6 to 36 h was probably due to the environmental disturbance by transferring worms from NGM agar pad to liquid medium. Moreover, the result that the untreated control showed that a time-dependent significant increase in germline cells apoptosis is due to maintenance of germline homeostasis (24, 25). Arsenite has been reported to inhibit mitotic division in a variety of cell types (15, 38, 40, 41). The perturbations of the spindle apparatus and tubulin were thought to be the mechanisms for mitotic disruption (42). Because the checkpoint pathways are evolutionally conserved, C. elegans germline cell cycle arrest induced by arsenic may share some common signal transduction pathways with other experimental animals. Various in vitro studies have shown that arsenite induces apoptosis in promyelocytic leukemia cells (43), chronic lymphocytic leukemia (BCLL) cells (5), and normal embryonic cells (15). However, the mechanisms of arsenic-induced apoptosis remain unknown, although caspase activation and enhanced generation of ROS have been postulated to be involved in the process (14, 44). Our present studies confirmed that arsenite increases ROS production in the intestine and not in gonad of C. elegans. This might be due to the nature of the fluorescence dye CM-H2DCFDA, which is nonpolar and can be converted by cellular esterases into a nonfluorescent membrane-impermeable polar derivative DCFH (45), which cannot reach the gonad and retained in the intestinal cells. Senoo-Matsuda et al. (46) demonstrated that C. elegans meV-1 mutant was hypersensitive to oxidative stress due to the overproduction of ROS in the meV-1 mutants. There is recent evidence to link mitochondrial ROS generation with the induction of nuclear DNA damage and subsequent mutagenesis of C. elegans fem-3 gene (47). The present data, which indicated that a 30 min arsenite exposure significantly increased ROS production in C. elegans, collaborated with these findings. The involvement of ROS in germline apoptosis and cell cycle arrest mediated by arsenic is further supported by the data obtained using the radical scavenger DMSO. On the other hand, there is evidence that suggests that activation of the JNK and/or p38 pathways may be important in arsenite-mediated apoptotic cell death (40, 48). Inoue et al. (35) reported that the activation p38 MAPK signaling pathway in C. elegans was required to activate the oxidative stress response induced by arsenite treatment. Our findings strongly supported the presumption that, in reaction to arsenic stress, the nematode C. elegans might share the same signal transduction pathways. In summary, exposing C. elegans to arsenite caused germline cell cycle arrest and apoptosis. The addition of DMSO rescued cell cycle arrest and suppressed apoptosis, indicating that ROS might involve in the damage process. Our present findings that C. elegans is sensitive to the genotoxic effects of arsenic suggest that C. elegans can be a sentinel marker for arsenic contamination of drinking water. Acknowledgment. This project was supported by the National Natural Science Foundation of China for Distinguished Young Scholars under Grants 10225526 and 2006Z026 and the

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Knowledge Innovation Program of the Chinese Academy of Sciences under Grant KSCX2-SW-324.

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