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Cd toxicity on lifespan of nematode Caenorhabditis elegans
Ecotoxicology and Environmental Safety 73 (2010) 1221–1230
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Formation of a combined Ca/Cd toxicity on lifespan of nematode Caenorhabditis elegans$, $$ Dayong Wang n, Peidang Liu, Yichao Yang, Lulu Shen Key Laboratory of Developmental Genes and Human Disease in Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing 210009, China
a r t i c l e in fo
abstract
Article history: Received 6 January 2010 Received in revised form 24 April 2010 Accepted 1 May 2010 Available online 30 June 2010
We investigated the possible formation of combined toxicity from Ca/Cd exposure on nematode lifespan. Ca exposure at concentrations more than 1.56 mM significantly reduced lifespan, accelerated aging-related declines, and induced severe stress response in wild-type nematodes. Combined Ca (25 mM)/Cd (200 mM) exposure decreased the lifespans compared to Cd (200 mM) exposure; whereas no lifespan differences were found between Ca (1.56 mM)/Cd (200 mM) exposure and Cd (200 mM) exposure. Combined Ca (25 mM)/Cd (200 mM) exposure caused a more significant induction of hsp-16.2::gfp expression, and a more severe increase in oxidative damage than Cd (200 mM) exposure. Moreover, mutation of mev-1, encoding a subunit of succinate dehydrogenase cytochrome b, enhanced the combined Ca/Cd toxicity on lifespan. Furthermore, mutation of daf-16, encoding a fork-head-family transcription factor, enhanced the combined Ca/Cd toxicity on lifespan, and mutation of daf-2, encoding an insulin receptor-like protein, alleviated the combined Ca/Cd toxicity on lifespan. & 2010 Elsevier Inc. All rights reserved.
Keywords: Lifespan Ca Ca/Cd Oxidative stress Combined toxicity Insulin signaling Caenorhabditis elegans
1. Introduction Although calcium (Ca) is an important secondary messenger in cells, excess deposition of Ca in cytosol will cause the cellular damage and even cell death. It was reported that the liver damage caused by toxic chemicals was closely associated with the accumulation of a large amount of calcium, and the calcium entry into the cell may be involved in tissue damage (Harman and Maxwell, 1995). Recently, the adverse effects from Ca exposure were further observed in nematode Caenorhabditis elegans. Exposure to a high level of Ca induced the severe toxicity on lifespan in nematodes, and the high levels of Ca, Al and Fe in a paper recycling mill effluent could account for the severe toxicity from the original effluent on lifespan of exposed nematodes (Wang et al., 2008b). So far, C. elegans has been chosen as a valuable bioindicator for toxicity tests because of its short life cycle, ease of generating mass cultures and low cost (Traunsperger et al., 1997; Mutwakil et al., 1997; Power and de Pomerai, 1999). By virtue of these
$ Funding source: This work was supported by the Grants from the National Natural Science Foundation of China (Nos. 30771113 and 30870810). $$ Assurance: Any study involving humans or experimental animals was conducted in accordance with national and institutional guidelines for the protection of human subjects and animal welfare. n Corresponding author. E-mail address: [email protected] (D. Wang).
0147-6513/$ - see front matter & 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2010.05.002
properties, C. elegans has been used widely for the ecological risk assessment and study of environmental toxicology (Roh et al., 2006; Leung et al., 2008; Wang and Xing, 2009). In addition, exposure to toxicants can often induce a severe elevation of stress response and the formation of oxidative stress in nematodes (Wang et al., 2007b; Wang and Wang, 2008b; Xiao et al., 2009). Nevertheless, the adverse effects from Ca exposure on nematode development are still largely unclear. Moreover, it is unknown whether the combined toxicity for Ca with other metals can be formed in nematodes. Animals’ lifespan can be influenced by environmental cues, and among these environmental cues, metal exposure will alter the lifespan to different degrees (Lin et al., 2006; Harada et al., 2007; Wang et al., 2007a, b; Wang and Wang, 2008a, b). The reduced lifespan can be usually observed in nematodes exposed to high levels of heavy metals (Harada et al., 2007; Wang et al., 2007a, b; Wang and Wang, 2008a, b). Thus, the first aim of this project was to investigate the possible effects of Ca exposure at different concentrations on lifespan, stress response and oxidative damage in nematodes. Moreover, considering the facts that the Cd toxicity has been well investigated in nematodes, and Cd exposure can also result in the severe decrease in nematode lifespan (Swain et al., 2004), the second aim was to examine the possible formation of combined toxicity from Ca/Cd exposure on lifespan in C. elegans. Furthermore, considering that the insulin signaling pathway is well studied in the nematodes for regulating lifespan (Braeckman and Vanfleteren, 2007), the third aim was to
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investigate the possible role of insulin signaling in regulating the formation of combined toxicity from Ca/Cd exposure on lifespan in C. elegans. Our data indicate the formation of combined toxicity from Ca/Cd exposure on nematode lifespan. In addition, our data strongly imply the important role of oxidative stress and insulin signaling in the formation of combined toxicity from Ca/Cd exposure on lifespan in C. elegans.
2. Materials and methods 2.1. Reagents Ca concentrations used in this study referred to the previous description (Wang et al., 2008b), and they were 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50 and 100 mM. The Cd concentration used was 225 mM, which was selected as described (Chu and Chow, 2002). Metal concentrations of exposed solutions were analyzed by atomic absorption spectrophotometry (AAS, Pye-Unicam model SP9, Cambridge, UK). The internal Ca level was measured using ICP-MS (inductively coupled plasma mass spectrometry) as described (Simpson et al., 2005; Lin et al., 2006). Exactly 40 nematodes were picked on day 7 of their adult life culture plates containing no added Ca or 0.78125–100 mM Ca into a microcentrifuge tube containing 150 ml deionized water. The tube was incubated for 2 h at 98 1C to dehydrate the nematodes. After dehydration followed by cooling, nematodes were digested at 98 1C for 18 h in 1 ml of 20% nitric acid, and then diluted to 3.6 ml for metal level measurement using a HP-4500 ICP-MS. A standard curve was generated as described (Simpson et al., 2005; Lin et al., 2006). A blank containing no nematodes was also prepared each time to assess the background levels of Ca ions introduced in the procedure. All the chemicals were obtained from SigmaAldrich (St. Louis, MO, USA). 2.2. Preparation of nematode cultures The strains used in the current study were wild-type N2, and mutants of mev-1(kn1), daf-16(mu86), daf-2(e1370), daf-16(mu86);daf-2(e1370);muEx169, and daf-16(mu86);daf-2(e1370);muEx211, originally obtained from the Caenorhabditis Genetics Center (funded by the NIH National Center for Research Resource, Minneapolis, MN, USA). KC136 (Ex(Phsp-16.2::gfp)) is the gift from Dr. King Chow’s lab. They were maintained on nematode growth medium (NGM) plates seeded with Escherichia coli OP50 at 20 1C as described (Brenner, 1974). Gravid nematodes were washed off the plates into centrifuge tubes, and were lysed with a bleaching mixture (0.45 M NaOH, 2% HOCl). Age synchronous populations of nematodes (L4-stage larvae) were obtained by the collection as described (Donkin and Williams, 1995). The examined nematodes were washed with double-distilled water twice, followed by washing with modified K medium once (50 mM NaCl, 30 mM KCl, 10 mM NaOAc, pH 5.5) (Williams and Dusenbery, 1990). Exposures were performed in 12-well sterile tissue culture plates as described (Mutwakil et al., 1997). All exposures were 48 h long and were carried out in 20 1C incubator in the presence of food. 2.3. Lifespan assay The lifespan assay was performed basically as described, and was initiated at 20 1C after metal exposure (Wilson et al., 2006; Shen et al., 2007). In this test, the hermaphrodites were transferred daily for the first 4 days of adulthood. The nematodes were checked every 2 day and would be scored as dead when they did not move even after repeated taps with a pick. For lifespan, graphs are representative of at least three trials. The data was statistically analyzed using a 2-tailed 2 sample t-test (Minitab Ltd., Coventry, UK). 2.4. Photography of autofluorescence Intestinal autofluorescence, caused by lysosomal deposits of lipofuscin, can accumulate over time in the aging nematodes and is a valuable marker for nematode aging (Boehm and Slack, 2005). The method for intestinal autofluorescence photography was performed as described (Garigan et al., 2002; Shen et al., 2007). The images were collected for endogenous intestine fluorescence using a 525 nm bandpass filter and without automatic gain control in order to preserve the relative intensity of different animal’s fluorescence. Day 4, day 8 and day 12 adults were photographed on the same day to avoid effects of light source variance on fluorescence intensity. Observations of the green fluorescent protein (GFP) were recorded and color images were taken for the documentation of results with Magnafires software (Olympus, Irving, TX, USA). Lipofuscin levels were measured using ImageJ Software (NIH Image) by determining average pixel intensity in each animal’s intestine. More than 50 animals were counted for the statistical analysis.
2.5. Analysis of transgenic strain The expression of HSP-16, a classical stress protein, can be induced by an array of environmental stresses including heavy metal exposure, heat-shock and oxidative stress (Chu and Chow, 2002; Wang et al., 2007b; Shen et al., 2009). It was reasoned that if the metal exposure is toxic, it would thus result in a stress response (Chu and Chow, 2002; Wang et al., 2007b; Wang and Wang, 2008b). The method was performed as previously described (Chu and Chow, 2002; Wang and Wang, 2008b). To analyze the changes of hsp-16.2 expression patterns, the treated KC136 animals were allowed to settle for 10 min, and then pipetted onto an agar pad on a glass slide, mounted and observed for the fluorescent signals with a fluorescent microscope. Observations of the GFP were recorded and color images were taken for the documentation of results with Magnafires software (Olympus, Irving, TX, USA). To distinguish the positive results from the background in the stress tested, only concentrations that could induce 50% signal were taken as having positive effects (Chu and Chow, 2002). More than 50 animals were counted for the statistical analysis. 2.6. Superoxide dismutase (SOD) activity The treated or control adult animals were used for the assay of SOD activities as described previously (Wang and Wang, 2008b). The SOD activity was measured using the kit from Randox Laboratories following the manufacturer’s protocol. The data were the summary of five trials. 2.7. Oxidative damage assay The collected gravid adult nematodes from ten 100 mm NGM plates were bleached to recover eggs, and the eggs were allowed to hatch in M9 buffer over a period of 5 days before L1 nematodes were transferred to NGM plates. At adulthood nematodes were collected in M9 buffer, washed free of bacteria, pelleted and frozen for carbonylated protein quantification. Proteins were quantified using a bicinchronic acid protein assay kit (Thermo Scientific) according to the manufacturer’s protocol. The data were the summary of five trials. 2.8. Brood size and body size assay The brood size and body size assay methods were performed as previously described (Wang and Wang, 2008a). Brood size was assayed by placing single tested nematode into individual well of tissue culture plates. The nematodes were transferred to a new well every 1.5 days. Progeny were counted the day following transfer, and at least 10 replicates were performed for the statistical purpose. The tested nematodes were picked out directly for body size measure. Body size was determined by measuring the flat surface area of nematodes using Image-Pros Express software. For each test, at least 30 nematodes were picked for assay. Three independent repeat experiments were performed. 2.9. Statistical analysis All data in this article were expressed as means7 SD. Graphs were generated using Microsoft Excel (Microsoft Corp., Redmond, WA). Analysis of variance (ANOVA) followed by a Dunnett’s t-test was used to determine the significance of the differences between the groups. The probability levels of 0.05 and 0.01 were considered statistically significant.
3. Results 3.1. Ca exposure at higher concentrations reduces the lifespan of wild-type N2 nematodes We first examined the effects of Ca exposure at different concentrations on the lifespan in adult wild-type N2 animals grown at 20 1C. As shown in Fig. 1, adult wild-type nematodes grown under our laboratory conditions had a mean lifespan of 17.5 days and an average maximum lifespan of 27.5 days at 20 1C. Exposure to 0.78 mM of Ca would not obviously affect the mean lifespan and maximum lifespan of treated wild-type nematodes. However, exposure to Ca at concentrations from 1.56 to 100 mM could obviously decrease the maximum lifespan of wild-type animals by 2.9%, 11.6%, 15.6%, 22.5%, 24.4%, 26.9% and 33.8%. Similarly, exposure to Ca at concentrations from 1.56 to 100 mM could significantly (1.56 mM, Po0.05; 3.13–100 mM, Po0.01) reduce the mean
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lifespan of wild-type animals by 12.6%, 15.4%, 17.7%, 21.1%, 31.4%, 38.9% and 50.9%. Moreover, we saw an obvious increase in the internal Ca levels in the exposed nematodes with the increased supplementation of Ca, suggesting that the Ca supplemented was taken up by the nematodes. Thus, Ca exposure at concentrations more than 1.56 mM will severely reduce lifespan of wild-type N2 nematodes. 3.2. The shortened lifespan in Ca exposed nematodes may be due to changed aging In order to validate the lifespan results, formation of lipofuscin was analyzed in Ca exposed nematodes. The lipofuscin is composed of cross-linked protein residues that irreversibly form due to oxidative processes (Brunk and Terman, 2002; Pluskota et al., 2009). To monitor the aging process, we examined the accumulation of intestinal autofluorescence in Ca exposed adult nematodes. As shown in Fig. 2, exposure to Ca at concentrations from 1.56 to 100 mM could induce a more rapid accumulation of intestinal autofluorescence than control at day 12 (Po0.01). Nematodes exposed to Ca at concentrations from 6.25 mM to 100 mM also displayed a remarkably increased lipofuscin-like intestinal autofluorescence at day 8 (6.25 mM, Po0.05; 12.5–100 mM, Po0.01). Moreover, early at day 4, nematodes exposed to Ca at concentrations from 25 mM to 100 mM could accumulate intestinal autofluorescence more rapidly than control (25 mM, Po0.05; 50–100 mM, Po0.01). These results were consistent with the lifespan analysis above, suggesting that the observed short-lived phenotype induced by Ca exposure may be due to the formation of age-related oxidative processes in C. elegans. 3.3. Ca exposure at higher concentrations induces the severe stress response Again, we explored the stable transgenic line of KC136 to investigate the possible stress response induced by Ca exposure at higher concentrations. Stress response was monitored by the significant induction of hsp-16.2::gfp expression (more than 50% of a population, above the line). As shown in Fig. 3, exposure to Ca at the concentration of 0.78 mM could not induce a severe stress response. In contrast to this, exposure to Ca at concentrations of 1.56 and 3.13 mM induced a moderate but significant (Po0.05) induction of hsp-16.2::gfp expression compared to control. Furthermore, nematodes exposed to Ca at concentrations more than 6.25 mM showed a very severe stress response as reflected by the noticeable (Po0.01) induction of hsp-16.2::gfp expression compared to control. This observation further suggests that Ca exposure at higher concentrations may induce the severe stress response in C. elegans. 3.4. Exposure to a high concentration of Ca with Cd induces the combinational toxicity on lifespan in wild-type N2 nematodes We next investigated the possible effects of combined Ca/Cd exposure on lifespan in wild-type N2 nematodes. As shown in Fig. 4, under our experimental conditions, exposure to 200 mM of Cd resulted in noticeably reduced maximum lifespan and mean lifespan compared to control. Combined exposure to Ca (25 mM)/ Cd (200 mM) could decrease the maximum lifespan by 15.9% compared to single exposure to Ca (25 mM), and by 17.5% compared to single exposure to Cd (200 mM), respectively; however, no difference could be found for maximum lifespan between combined exposure to Ca (1.56 mM)/Cd (200 mM) and single exposure to Cd (200 mM). Similarly, combined exposure to
Fig. 1. Effects of Ca exposure on lifespan in wild-type N2 nematodes. (A) Effects of Ca exposure at different concentrations on lifespan of nematodes grown at 20 1C. (B) Comparison of mean lifespans in nematodes exposed to Ca at different concentrations. (C) Internal Ca concentration determination using ICP-MS. Data are expressed as mean 7 SD. nPo 0.05 vs. 0 mM, nnP o0.01 vs. 0 mM.
Ca (25 mM)/Cd (200 mM) could reduce the mean lifespan by 40% compared to single exposure to Ca (25 mM), and by 25% compared to single exposure to Cd (200 mM), respectively. No differences could be observed for mean lifespan between combined exposure to Ca (1.56 mM)/Cd (200 mM) and single exposure to Cd (200 mM). These results demonstrate that the combined exposure to a high concentration (25 mM) of Ca with Cd will induce an obviously synergistic toxicity on lifespan in nematodes.
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Ca (1.56 mM)/Cd (200 mM) was similar to that in nematodes exposed to Cd (200 mM). Therefore, the exposure to high level of Ca may be required for inducing the obviously combined Ca/Cd toxicity on lifespan by activating more severe stress response in nematodes. 3.6. Combined exposure to a high concentration of Ca with Cd induces more severe oxidative stress than single metal exposure in wild-type N2 nematodes
Fig. 2. Effects of Ca exposure at different concentrations on mean autofluorescence from intestine lipofuscin in day 4, day 8 and day 12 adult nematodes. Data are expressed as mean 7SD. nPo 0.05 vs. 0 mM, nnP o 0.01 vs. 0 mM.
Fig. 3. Effects of Ca exposure on stress responses in wild-type N2 nematodes. Significant induction of hsp-16.2::gfp expression (more than 50% of a population, above the line) was observed in wild-type N2 nematodes exposed to Ca at concentrations more than 1.56 mM compared to control. Data are expressed as mean 7 SD. nP o 0.05 vs. 0 mM, nnPo 0.01 vs. 0 mM.
We further ask whether the induction of combined Ca/Cd toxicity on lifespan is enhancing the damage from oxidative stress. SOD belongs to the major defense enzymes against superoxide radicals, and its activities are directly linked to oxidative stress (Yanase et al., 2002; Wang and Wang, 2008b). As shown in Fig. 6A and B, under our experimental conditions, exposure to Ca at concentrations of 25 mM (Po0.01) and 1.56 mM (Po0.05), and to Cd at the concentration of 200 mM (Po 0.01) all significantly decreased the SOD activities in exposed nematodes compared to control. Combined exposure to Ca (25 mM)/Cd (200 mM) could cause a more severe reduction in SOD activity than single exposure to Ca (25 mM) or to Cd (200 mM), whereas the SOD activity in nematodes exposed to Ca (1.56 mM)/Cd (200 mM) was similar to that in nematodes exposed to Cd (200 mM). The observations above were further confirmed by the analysis of oxidative damage. As shown in Fig. 6C and D, exposure to Ca at concentrations of 25 (Po0.01) and 1.56 mM (P o0.05), and to Cd at the concentration of 200 mM (Po0.01) all significantly increased the oxidative damage in exposed nematodes compared to control. Furthermore, combined exposure to Ca (25 mM)/ Cd (200 mM) induced a more severe increase in oxidative damage in exposed nematodes than single exposure to Ca (25 mM) or to Cd (200 mM); however, no significant alterations of oxidative damage could be detected in nematodes exposed to Ca (1.56 mM)/ Cd (200 mM) from those in nematodes exposed to Cd (200 mM). Therefore, combined exposure to a high concentration (25 mM) of Ca with Cd may induce the synergistic toxicity on lifespan by activating more severe oxidative stress in nematodes. 3.7. Combinational Ca/Cd toxicity on lifespan is dependent of the expression of mev-1
To examine whether the effect of 1.56 mM Ca may be masked by the strong effect of Cd, we further selected an intermediate range of Ca (12.5 mM) to investigate the combined exposure to Ca (12.5 mM)/Cd (200 mM) on lifespan, and combined exposure to Ca (12.5 mM)/Cd (200 mM) could noticeably decrease the lifespan compared to single exposure to Ca (12.5 mM) or to Cd (200 mM). These data imply that Ca at 1.56 mM may still have effect with Cd at 200 mM, but the difference was not detectable in our assay.
3.5. Combined exposure to a high concentration of Ca with Cd activates more severe stress response than single metal exposure in wild-type N2 nematodes We next determined the possible effects of combined exposure to a high concentration of Ca with Cd on stress response. As shown in Fig. 5, exposure to 200 mM of Cd would cause a noticeable induction of hsp-16.2::gfp expression (more than 50% of a population, above the line) compared to control. Moreover, combined exposure to Ca (25 mM)/Cd (200 mM) resulted in a more significant induction of hsp-16.2::gfp expression than single exposure to Ca (25 mM) or to Cd (200 mM). Different from this, the induction of hsp-16.2::gfp expression in nematodes exposed to
The gene mev-1 encodes a subunit of the enzyme succinate dehydrogenase cytochrome b, which is a component of complex II of the mitochondrial electron transport chain (Ishii et al., 1998). mev-1 governs the rate of ageing by modulating the cellular response to oxidative stress in C. elegans (Ishii et al., 1998). The mev-1 mutant is significantly short-lived with several phenotypes consistent with elevated oxidative stress (Lin et al., 2006). Again, we investigated the effects of mev-1 mutation on the formation of combined Ca/Cd toxicity on lifespan in nematodes. As shown in Fig. 7, exposure to Ca (25 mM) or Cd (200 mM) more noticeably reduced the maximum and mean lifespans in mev-1 mutant nematodes than those in wild-type N2 nematodes. In addition, both the maximum lifespan and mean lifespan in Ca (25 mM)/ Cd (200 mM) exposed mev-1 mutant nematodes were shorter that those in Ca (25 mM)/Cd (200 mM) exposed wild-type N2 nematodes. Moreover, mutation of mev-1 could not block the formation of combinational Ca/Cd toxicity on lifespan. Nevertheless, mutation of mev-1 obviously enhanced the combined Ca/Cd toxicity on lifespan in nematodes. Under our experimental conditions, combined exposure to Ca (25 mM)/Cd (200 mM) could decrease the mean lifespan by approximately 2.4 days compared to single exposure to Cd (200 mM) in wild-type N2 nematodes. In contrast,
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Fig. 4. Effects of combined Ca/Cd exposure on lifespan in wild-type N2 nematodes. (A) Effects of combined exposure of Ca at the concentration of 25 mM with Cd (200 mM) on lifespan of nematodes grown at 20 1C. (B) Effects of combined exposure of Ca at the concentration of 1.56 mM with Cd (200 mM) on lifespan of nematodes grown at 20 1C. (C) Effects of combined exposure of Ca at the concentration of 12.5 mM with Cd (200 mM) on lifespan of nematodes grown at 20 1C. (D) Combined exposure with Ca (25 mM)/Cd (200 mM) induced a more decreased lifespan than single exposure to Ca (25 mM) or Cd (200 mM). (E) Combined exposure with Ca (1.56 mM)/Cd (200 mM) induced a similarly decreased lifespan to single exposure to Cd (200 mM). (F) Combined exposure with Ca (12.5 mM)/Cd (200 mM) induced a more decreased lifespan than single exposure to Cd (200 mM). Control, without metal exposure. NS, no significant difference. Data are expressed as mean 7 SD. nnPo 0.01.
combined exposure to Ca (25 mM)/Cd (200 mM) reduced the mean lifespan by approximately 4.1 days compared to single exposure to Cd (200 mM) in mev-1 mutant nematodes, suggesting that the formation of combinational Ca/Cd toxicity on lifespan is dependent on the expression of mev-1.
3.8. Insulin signaling pathway regulates the formation of synergistic Ca/Cd toxicity on lifespan in nematodes In C. elegans, the lifespan is regulated hormonally by an insulin/IGF-like signaling pathway. In DAF-2 pathway mutants,
DAF-16, a fork-head-family transcription factor, accumulates in the nuclei of many cell types, where it results in changes in the expression of a wide variety of response, and thereby extends lifespan (Braeckman and Vanfleteren, 2007). Previous study has demonstrated that mutation of daf-2 can result in increased metal resistance (Barsyte et al., 2001). To examine whether the insulin signaling pathway is involved in the regulation of synergistic Ca/Cd toxicity on lifespan, we next investigated the effects of daf-16 and daf-2 mutation on the formation of synergistic Ca/Cd toxicity on lifespan in nematodes. The mean lifespan in Ca (25 mM)/Cd (200 mM) exposed daf-16 mutant nematodes was shorter than those in Ca (25 mM)/Cd (200 mM) exposed
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Fig. 5. Effects of combined Ca/Cd exposure on stress responses in wild-type N2 nematodes. (A) Effects of combined exposure of Ca at the concentration of 25 mM with Cd (200 mM) on the induction of hsp-16.2::gfp expression. (B) Effects of combined exposure of Ca at the concentration of 1.56 mM with Cd (200 mM) on the induction of hsp-16.2::gfp expression. Stress response was monitored by the significant induction of hsp-16.2::gfp expression (more than 50% of a population, above the line). Control, without metal exposure. NS, no significant difference. Data are expressed as mean 7 SD. nnP o 0.01.
Fig. 6. Effects of combined Ca/Cd exposure on superoxide dismutase (SOD) activity, and oxidative damage in wild-type N2 nematodes. (A) Effects of combined exposure of Ca at the concentration of 25 mM with Cd (200 mM) on SOD activity. (B) Effects of combined exposure of Ca at the concentration of 1.56 mM with Cd (200 mM) on SOD activity. (C) Effects of combined exposure of Ca at the concentration of 25 mM with Cd (200 mM) on oxidative damage. (D) Effects of combined exposure of Ca at the concentration of 1.56 mM with Cd (200 mM) on oxidative damage. Control, without metal exposure. NS, no significant difference. Data are expressed as mean 7 SD. n Po 0.05 vs. control, nnPo 0.01 vs. control (if not specially indicated).
wild-type N2 nematodes (Fig. 8A). In contrast, the mean lifespan in Ca (25 mM)/Cd (200 mM) exposed daf-2 mutant nematodes was longer than those in Ca (25 mM)/Cd (200 mM) exposed wild-type N2 nematodes (Fig. 8B). In addition, mutation of daf-16 and daf-2 could not block the formation of synergistic Ca/Cd toxicity on nematode longevity (Fig. 8A and B). Nevertheless, mutation of daf-16 could obviously enhance the synergistic Ca/Cd toxicity on nematode longevity, but mutation of daf-2 could noticeably alleviate the synergistic Ca/Cd toxicity on nematode longevity.
Under our experimental conditions, combined exposure to Ca (25 mM)/Cd (200 mM) reduced the mean lifespan by approximately 3.6 days compared to single exposure to Cd (200 mM) in daf-16 mutant nematodes (Fig. 8A), and combined exposure to Ca (25 mM)/Cd (200 mM) reduced the mean lifespan by approximately 1.4 days compared to single exposure to Cd (200 mM) in daf-2 mutant nematodes (Fig. 8B). These data suggest that the formation of synergistic Ca/Cd toxicity on lifespan is at least partially dependent of the insulin signaling.
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In C. elegans, DAF-16 activities in the intestine and nervous system could completely or significantly restore the longevity of daf-16(-) germline-deficient nematodes, and increase the lifespans of daf-16(-) insulin/IGF-1-pathway mutants substantially (Libina et al., 2003). We further examined the tissue-specific activities of DAF-16 in the regulation of the synergistic Ca/Cd toxicity on lifespan of nematodes. The effects of Ca (25 mM)/ Cd (200 mM) exposure on the lifespan of daf-16;daf-2 mutant nematodes were similar to those on the lifespan of wild-type N2 nematodes (data not shown). The mean lifespan in Ca (25 mM)/ Cd (200 mM) exposed daf-16;daf-2;muEx211/Pges-1::gfp::daf-16 and daf-16;daf-2;muEx169/Punc-119::gfp::daf-16 nematodes was longer than those in Ca (25 mM)/Cd (200 mM) exposed wild-type N2 nematodes (Fig. 8C and D). Similarly, over-expression of intestinal DAF-16 (Pges-1::gfp::daf-16) and neuronal DAF-16 (Punc-119::gfp::daf-16) could not block the formation of synergistic Ca/Cd toxicity on lifespan of daf-16;daf-2 mutant nematodes (Fig. 8C and D). Nevertheless, over-expression of intestinal and neuronal DAF-16 could significantly alleviate the synergistic Ca/Cd toxicity on lifespan of nematodes. Combined exposure to Ca (25 mM)/Cd (200 mM) reduced the mean lifespan by approximately 1.1 days compared to single exposure to Cd (200 mM) in
Fig. 7. Effects of combined Ca/Cd exposure on lifespan of mev-1 nematode mutant. (A) Effects of combined exposure of Ca at the concentration of 25 mM with Cd (200 mM) on lifespan in wild-type N2 and mev-1 mutant nematodes grown at 20 1C. (B) Mean lifespan comparison in wild-type N2 and mev-1 mutant nematodes. Data are expressed as mean 7 SD. nPo 0.05 vs. mev-1, nnP o 0.01 vs. N2 or mev-1 (if not specially indicated).
Fig. 8. Effects of insulin signaling on the formation of synergistic Ca/Cd toxicity on lifespan in nematodes. (A) Effects of combined exposure of Ca at the concentration of 25 mM with Cd (200 mM) on mean lifespan in wild-type N2 and daf-16 mutant nematodes grown at 20 1C. (B) Effects of combined exposure of Ca at the concentration of 25 mM with Cd (200 mM) on mean lifespan in wild-type N2 and daf-2 mutant nematodes grown at 20 1C. (C) Effects of combined exposure of Ca at the concentration of 25 mM with Cd (200 mM) on mean lifespan in wild-type N2 and daf-16;daf-2;muEx211 mutant nematodes grown at 20 1C. (D) Effects of combined exposure of Ca at the concentration of 25 mM with Cd (200 mM) on mean lifespan in wild-type N2 and daf-16;daf-2;muEx169 mutant nematodes grown at 20 1C. Data are expressed as mean 7SD. nP o 0.05 vs. wild-type or mutant (if not specially indicated), nnP o0.01 vs. wild-type or mutant (if not specially indicated).
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daf-16;daf-2;muEx211 mutant nematodes (Fig. 8C), and combined exposure to Ca (25 mM)/Cd (200 mM) reduced the mean lifespan by approximately 1.8 days compared to single exposure to Cd
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Fig. 9. Effects of metal exposure on brood size (A) and body size (B). Data are expressed as mean 7 SD. nP o 0.05 vs. control,
(200 mM) in daf-16;daf-2;muEx169 mutant nematodes (Fig. 8D). Therefore, the intestinal and neuronal DAF-16 activities are involved in the control of synergistic Ca/Cd toxicity lifespan of nematodes. 3.9. Effects of Ca and combined Ca/Cd exposure on brood size and nematode development We finally investigated the effects of Ca and combined Ca/Cd exposure on brood size and development in nematodes. As shown in Fig. 9, exposure to Ca at concentrations from 0.78 to 25 mM could not cause obvious decrease in brood size and body size, whereas the noticeable decrease in brood size or body size could be observed in nematodes exposed to 50 and 100 mM of Ca. In addition, exposure to Cd (200 mM) and combined Ca (25 mM)/ Cd (200 mM) could also result in significant decreases in brood size and body size compared to control. Moreover, no obvious differences could be found for brood size and body size in nematodes exposed to Cd (200 mM) from those in nematodes exposed to Ca (25 mM)/Cd (200 mM). Furthermore, exposure to Ca at different concentrations and combined Ca (25 mM)/ Cd (200 mM) could not induce severe deficits in dauer formation compared to control (data not shown).
4. Discussion In the present study, we first provide the evidence to indicate the important roles of Ca exposure at different concentrations in influencing the lifespan of nematodes. Ca exposure at concentrations more than 1.56 mM can induce the severely reduced lifespan in wild-type N2 nematodes (Fig. 1). In addition, the analysis on the accumulation of intestinal autofluorescence suggested that the observed short-lived nematodes appeared after Ca exposure may be due to accelerated aging, and not due to an unrelated, pleiotropic cause (Fig. 2). These conclusions on the Ca toxicity on lifespan in nematodes are largely consistent with the observations in other animals and even in plants. The average lifespan of the rotifer Asplanchna brightwelli could be significantly reduced when 1.2 or 2.4 mM CaCl2 was added to the culture medium, and the maximum lifespan was also reduced as the CaCl2 concentration was increased (Enesco and Holtzman, 1980). Studies on the function of PPF1 during plant development further suggest that internal Ca2 + concentration or calcium storage capacity is closely related to the induction of cell death or cell senescence in plant cells (Li et al., 2004; Wang et al., 2008a).
P o 0.01 vs. control.
nn
The contribution of the release of calcium from smooth endoplasmic reticulum calcium stores can also be altered with age in peripheral neurons (Buchholz et al., 2007). In addition, the exposure of hippocampal neurons to Ab peptides led to dendritic dystrophy and activation of apoptotic neuronal death that may be associated with a significant rise in cytosolic Ca2 + concentrations (Resende et al., 2007). In contrast, inhibition of Ca2 + influx during arsenic treatment reduced the cell death in Leishnania spp. (Mehta and Shaha, 2006). Similarly, simultaneous treatment with either verapamil (an inhibitor of Ca2 + through plasma membrane) or dantrolene (an inhibitor of mobilization of [Ca2 + ]i from endoplasmic reticulum) or BAPTA (a Ca2 + chelator) could obviously reduce Ni compound-induced DNA single-strand breaks in both chromosomal and nuclear chromatin (M’Bemba-Meka et al., 2005). Previous study has demonstrated that the synthetic SOD/CAT mimetics (SCMs), Euk-134 and Euk-8 confer resistance to the oxidative stress-inducing agent, paraquat and to thermal stress, and the lifespan of nematodes can be extended by the administration of Euk-134 and Euk-8 without any toxic effects on development and fertility, suggesting the involvement of oxidative stress in regulating nematode lifespan (Sampayo et al., 2003). The cellular Ca2 + levels hold the key to activating many response pathways, and high [Ca2 + ]i can cause disruption of mitochondrial Ca2 + equilibrium, which will result in reactive oxygen species (ROS) formation due to the stimulation of electron flux along the electron transport chain (Chacon and Acosta, 1991). In the current work, our data further suggest that Ca exposure at higher concentrations results in the severe stress response as monitored by the significantly elevated induction of hsp-16.2::gfp expression (Fig. 3). Moreover, the severe oxidative stress could be induced by the Ca exposure at higher concentrations as revealed by the decreased SOD activities, and the oxidative damage in C. elegans (Fig. 6). It was also reported that exposure to metals, such as Cu and Ba, may usually stimulate the formation of oxidative stress in animal cells (Nawaz et al., 2006; Wang and Wang, 2008b). Our data in this project further indicate the combined Ca/Cd toxicity on lifespan in C. elegans. Combined exposure to Ca (25 mM)/Cd (200 mM) could significantly decrease the maximum and mean lifespans compared to single exposure to Cd (200 mM); however, no difference could be found for maximum lifespan, and mean lifespan between combined exposure to Ca (1.56 mM)/Cd (200 mM) and single exposure to Cd (200 mM) (Fig. 4), suggesting that combined exposure to a high concentration of Ca with Cd can induce the combined toxicity on lifespan in wild-type N2 nematodes. Similarly, the combined Ca/Cd toxicity on lifespan
D. Wang et al. / Ecotoxicology and Environmental Safety 73 (2010) 1221–1230
could be observed in exposed planarians (Wang et al., personal communication). It was also reported that the copper toxicity increased at higher Ca:Mg ratios for Daphnia magna (Naddy et al., 2002). The main reason to select the Cd for the assay of combined toxicity on lifespan is that the toxicology from Cd exposure is the most well studied so far in C. elegans (Swain et al., 2004). At least four lines of reasoning made us select the Ca (25 mM) to perform the study on combinational Ca/Cd toxicity on nematode lifespan. First, exposure to 25 mM of Ca obviously reduced the maximum and mean lifespans of wild-type N2 nematodes (Fig. 1). Second, exposure to 25 mM of Ca could cause severe stress response (Fig. 3) and oxidative stress (Fig. 6). Third, exposure to 25 mM of Ca did not obviously alter the nematodes’ dauer formation (data not shown), brood size and body size (Fig. 9). Fourth, Ca (25 mM) exposure would not influence the dauer formation (data not shown), brood size and body size (Fig. 9) of nematodes exposed to Cd (200 mM). For the regulation mechanisms explaining the formation of combined Ca/Cd toxicity on nematode lifespan, we raised the notion that one of the mechanisms is the possible involvement of oxidative stress in the regulation of combined Ca/Cd toxicity on nematode lifespan. Combined exposure to Ca (25 mM)/ Cd (200 mM) could result in a more significant induction of hsp-16.2::gfp expression, a more severe reduction of SOD activity, and a more severe increase in oxidative damage than single exposure to Cd (200 mM) (Figs. 5 and 6), suggesting that combined exposure to a high concentration (25 mM) of Ca with Cd may cause the synergistic toxicity on lifespan by inducing more severe oxidative stress in nematodes. Moreover, because mutation of mev-1 could obviously enhance the synergistic Ca/Cd toxicity on nematode lifespan, the formation of combined Ca/Cd toxicity on lifespan is dependent of the expression of mev-1 (Fig. 7). Nevertheless, mutation of mev-1 could not block the formation of combined Ca/Cd toxicity on lifespan (Fig. 7). Similarly, induction of oxidative stress at early L2-larval stage by paraquat (2 mM) treatment could not also block the formation of combined Ca/Cd toxicity on lifespan (data not shown). These observations suggest that occurrence of severe oxidative stress may still not be the only mechanism to explain the formation of combined Ca/Cd toxicity on nematode lifespan. Another possible mechanism we raised to explain the formation of combined Ca/Cd toxicity on lifespan is the involvement of insulin signaling pathway. In the present study, our data suggest that the formation of combined Ca/Cd toxicity on lifespan is at least partially dependent on the insulin signaling, since mutation of daf-16 obviously enhanced the combined Ca/Cd toxicity on lifespan and mutation of daf-2 noticeably alleviated the combined Ca/Cd toxicity on lifespan (Fig. 8). Moreover, because overexpression of intestinal and neuronal DAF-16 could significantly alleviate the combined Ca/Cd toxicity on lifespan of daf-16;daf-2 mutant nematodes (Fig. 8), and over-expression of muscle DAF-16 had no noticeable effects on the formation of combined Ca/Cd toxicity on lifespan of daf-16;daf-2 mutant nematodes (data not shown), the intestinal and neuronal DAF-16 activities may be involved in the control of combined Ca/Cd toxicity on lifespan in C. elegans. Nevertheless, mutation of daf-16 and daf-2, as well as the over-expression of intestinal and neuronal DAF-16, could not block the formation of combined Ca/Cd toxicity on lifespan, which implies that multiple signaling pathways may regulate the formation of combined Ca/Cd toxicity on lifespan of nematodes. Because both the exposure to Ca (25 mM) and the exposure to Ca (25 mM)/Cd (200 mM) could not induce severe deficits in dauer formation compared to control (data not shown), so at least two signaling pathways, oxidative stress and insulin signaling pathway, may play important roles in regulating the formation of combined Ca/Cd toxicity on lifespan in C. elegans.
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5. Conclusions In conclusion, Ca exposure at concentrations more than 1.56 mM induces the severely reduced lifespan in wild-type N2 nematodes. Combined exposure to Ca (25 mM)/Cd (200 mM) significantly decreases the lifespans compared to single exposure to Cd (200 mM). Moreover, combined exposure to Ca (25 mM)/ Cd (200 mM) results in a more significant induction of hsp16.2::gfp expression, a more severe reduction in SOD activity, and a more severe increase in oxidative damage than single exposure to Cd (200 mM). Furthermore, both the oxidative stress and the insulin signaling pathway are involved in regulating the formation of combined Ca/Cd toxicity on lifespan in C. elegans.
Acknowledgments Strains used in this study were provided by the Caenorhabditis Genetics Center (Funded by the NIH, National Center for Foundation from Research Resource, USA). This work was supported by the National Natural Science Foundation of China (nos. 30771113 and 30870810).
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Formation of a combined Ca/Cd toxicity on lifespan of nematode Caenorhabditis elegans$, $$ Dayong Wang n, Peidang Liu, Yichao Yang, Lulu Shen Key Laboratory of Developmental Genes and Human Disease in Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing 210009, China
a r t i c l e in fo
abstract
Article history: Received 6 January 2010 Received in revised form 24 April 2010 Accepted 1 May 2010 Available online 30 June 2010
We investigated the possible formation of combined toxicity from Ca/Cd exposure on nematode lifespan. Ca exposure at concentrations more than 1.56 mM significantly reduced lifespan, accelerated aging-related declines, and induced severe stress response in wild-type nematodes. Combined Ca (25 mM)/Cd (200 mM) exposure decreased the lifespans compared to Cd (200 mM) exposure; whereas no lifespan differences were found between Ca (1.56 mM)/Cd (200 mM) exposure and Cd (200 mM) exposure. Combined Ca (25 mM)/Cd (200 mM) exposure caused a more significant induction of hsp-16.2::gfp expression, and a more severe increase in oxidative damage than Cd (200 mM) exposure. Moreover, mutation of mev-1, encoding a subunit of succinate dehydrogenase cytochrome b, enhanced the combined Ca/Cd toxicity on lifespan. Furthermore, mutation of daf-16, encoding a fork-head-family transcription factor, enhanced the combined Ca/Cd toxicity on lifespan, and mutation of daf-2, encoding an insulin receptor-like protein, alleviated the combined Ca/Cd toxicity on lifespan. & 2010 Elsevier Inc. All rights reserved.
Keywords: Lifespan Ca Ca/Cd Oxidative stress Combined toxicity Insulin signaling Caenorhabditis elegans
1. Introduction Although calcium (Ca) is an important secondary messenger in cells, excess deposition of Ca in cytosol will cause the cellular damage and even cell death. It was reported that the liver damage caused by toxic chemicals was closely associated with the accumulation of a large amount of calcium, and the calcium entry into the cell may be involved in tissue damage (Harman and Maxwell, 1995). Recently, the adverse effects from Ca exposure were further observed in nematode Caenorhabditis elegans. Exposure to a high level of Ca induced the severe toxicity on lifespan in nematodes, and the high levels of Ca, Al and Fe in a paper recycling mill effluent could account for the severe toxicity from the original effluent on lifespan of exposed nematodes (Wang et al., 2008b). So far, C. elegans has been chosen as a valuable bioindicator for toxicity tests because of its short life cycle, ease of generating mass cultures and low cost (Traunsperger et al., 1997; Mutwakil et al., 1997; Power and de Pomerai, 1999). By virtue of these
$ Funding source: This work was supported by the Grants from the National Natural Science Foundation of China (Nos. 30771113 and 30870810). $$ Assurance: Any study involving humans or experimental animals was conducted in accordance with national and institutional guidelines for the protection of human subjects and animal welfare. n Corresponding author. E-mail address: [email protected] (D. Wang).
0147-6513/$ - see front matter & 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2010.05.002
properties, C. elegans has been used widely for the ecological risk assessment and study of environmental toxicology (Roh et al., 2006; Leung et al., 2008; Wang and Xing, 2009). In addition, exposure to toxicants can often induce a severe elevation of stress response and the formation of oxidative stress in nematodes (Wang et al., 2007b; Wang and Wang, 2008b; Xiao et al., 2009). Nevertheless, the adverse effects from Ca exposure on nematode development are still largely unclear. Moreover, it is unknown whether the combined toxicity for Ca with other metals can be formed in nematodes. Animals’ lifespan can be influenced by environmental cues, and among these environmental cues, metal exposure will alter the lifespan to different degrees (Lin et al., 2006; Harada et al., 2007; Wang et al., 2007a, b; Wang and Wang, 2008a, b). The reduced lifespan can be usually observed in nematodes exposed to high levels of heavy metals (Harada et al., 2007; Wang et al., 2007a, b; Wang and Wang, 2008a, b). Thus, the first aim of this project was to investigate the possible effects of Ca exposure at different concentrations on lifespan, stress response and oxidative damage in nematodes. Moreover, considering the facts that the Cd toxicity has been well investigated in nematodes, and Cd exposure can also result in the severe decrease in nematode lifespan (Swain et al., 2004), the second aim was to examine the possible formation of combined toxicity from Ca/Cd exposure on lifespan in C. elegans. Furthermore, considering that the insulin signaling pathway is well studied in the nematodes for regulating lifespan (Braeckman and Vanfleteren, 2007), the third aim was to
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investigate the possible role of insulin signaling in regulating the formation of combined toxicity from Ca/Cd exposure on lifespan in C. elegans. Our data indicate the formation of combined toxicity from Ca/Cd exposure on nematode lifespan. In addition, our data strongly imply the important role of oxidative stress and insulin signaling in the formation of combined toxicity from Ca/Cd exposure on lifespan in C. elegans.
2. Materials and methods 2.1. Reagents Ca concentrations used in this study referred to the previous description (Wang et al., 2008b), and they were 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50 and 100 mM. The Cd concentration used was 225 mM, which was selected as described (Chu and Chow, 2002). Metal concentrations of exposed solutions were analyzed by atomic absorption spectrophotometry (AAS, Pye-Unicam model SP9, Cambridge, UK). The internal Ca level was measured using ICP-MS (inductively coupled plasma mass spectrometry) as described (Simpson et al., 2005; Lin et al., 2006). Exactly 40 nematodes were picked on day 7 of their adult life culture plates containing no added Ca or 0.78125–100 mM Ca into a microcentrifuge tube containing 150 ml deionized water. The tube was incubated for 2 h at 98 1C to dehydrate the nematodes. After dehydration followed by cooling, nematodes were digested at 98 1C for 18 h in 1 ml of 20% nitric acid, and then diluted to 3.6 ml for metal level measurement using a HP-4500 ICP-MS. A standard curve was generated as described (Simpson et al., 2005; Lin et al., 2006). A blank containing no nematodes was also prepared each time to assess the background levels of Ca ions introduced in the procedure. All the chemicals were obtained from SigmaAldrich (St. Louis, MO, USA). 2.2. Preparation of nematode cultures The strains used in the current study were wild-type N2, and mutants of mev-1(kn1), daf-16(mu86), daf-2(e1370), daf-16(mu86);daf-2(e1370);muEx169, and daf-16(mu86);daf-2(e1370);muEx211, originally obtained from the Caenorhabditis Genetics Center (funded by the NIH National Center for Research Resource, Minneapolis, MN, USA). KC136 (Ex(Phsp-16.2::gfp)) is the gift from Dr. King Chow’s lab. They were maintained on nematode growth medium (NGM) plates seeded with Escherichia coli OP50 at 20 1C as described (Brenner, 1974). Gravid nematodes were washed off the plates into centrifuge tubes, and were lysed with a bleaching mixture (0.45 M NaOH, 2% HOCl). Age synchronous populations of nematodes (L4-stage larvae) were obtained by the collection as described (Donkin and Williams, 1995). The examined nematodes were washed with double-distilled water twice, followed by washing with modified K medium once (50 mM NaCl, 30 mM KCl, 10 mM NaOAc, pH 5.5) (Williams and Dusenbery, 1990). Exposures were performed in 12-well sterile tissue culture plates as described (Mutwakil et al., 1997). All exposures were 48 h long and were carried out in 20 1C incubator in the presence of food. 2.3. Lifespan assay The lifespan assay was performed basically as described, and was initiated at 20 1C after metal exposure (Wilson et al., 2006; Shen et al., 2007). In this test, the hermaphrodites were transferred daily for the first 4 days of adulthood. The nematodes were checked every 2 day and would be scored as dead when they did not move even after repeated taps with a pick. For lifespan, graphs are representative of at least three trials. The data was statistically analyzed using a 2-tailed 2 sample t-test (Minitab Ltd., Coventry, UK). 2.4. Photography of autofluorescence Intestinal autofluorescence, caused by lysosomal deposits of lipofuscin, can accumulate over time in the aging nematodes and is a valuable marker for nematode aging (Boehm and Slack, 2005). The method for intestinal autofluorescence photography was performed as described (Garigan et al., 2002; Shen et al., 2007). The images were collected for endogenous intestine fluorescence using a 525 nm bandpass filter and without automatic gain control in order to preserve the relative intensity of different animal’s fluorescence. Day 4, day 8 and day 12 adults were photographed on the same day to avoid effects of light source variance on fluorescence intensity. Observations of the green fluorescent protein (GFP) were recorded and color images were taken for the documentation of results with Magnafires software (Olympus, Irving, TX, USA). Lipofuscin levels were measured using ImageJ Software (NIH Image) by determining average pixel intensity in each animal’s intestine. More than 50 animals were counted for the statistical analysis.
2.5. Analysis of transgenic strain The expression of HSP-16, a classical stress protein, can be induced by an array of environmental stresses including heavy metal exposure, heat-shock and oxidative stress (Chu and Chow, 2002; Wang et al., 2007b; Shen et al., 2009). It was reasoned that if the metal exposure is toxic, it would thus result in a stress response (Chu and Chow, 2002; Wang et al., 2007b; Wang and Wang, 2008b). The method was performed as previously described (Chu and Chow, 2002; Wang and Wang, 2008b). To analyze the changes of hsp-16.2 expression patterns, the treated KC136 animals were allowed to settle for 10 min, and then pipetted onto an agar pad on a glass slide, mounted and observed for the fluorescent signals with a fluorescent microscope. Observations of the GFP were recorded and color images were taken for the documentation of results with Magnafires software (Olympus, Irving, TX, USA). To distinguish the positive results from the background in the stress tested, only concentrations that could induce 50% signal were taken as having positive effects (Chu and Chow, 2002). More than 50 animals were counted for the statistical analysis. 2.6. Superoxide dismutase (SOD) activity The treated or control adult animals were used for the assay of SOD activities as described previously (Wang and Wang, 2008b). The SOD activity was measured using the kit from Randox Laboratories following the manufacturer’s protocol. The data were the summary of five trials. 2.7. Oxidative damage assay The collected gravid adult nematodes from ten 100 mm NGM plates were bleached to recover eggs, and the eggs were allowed to hatch in M9 buffer over a period of 5 days before L1 nematodes were transferred to NGM plates. At adulthood nematodes were collected in M9 buffer, washed free of bacteria, pelleted and frozen for carbonylated protein quantification. Proteins were quantified using a bicinchronic acid protein assay kit (Thermo Scientific) according to the manufacturer’s protocol. The data were the summary of five trials. 2.8. Brood size and body size assay The brood size and body size assay methods were performed as previously described (Wang and Wang, 2008a). Brood size was assayed by placing single tested nematode into individual well of tissue culture plates. The nematodes were transferred to a new well every 1.5 days. Progeny were counted the day following transfer, and at least 10 replicates were performed for the statistical purpose. The tested nematodes were picked out directly for body size measure. Body size was determined by measuring the flat surface area of nematodes using Image-Pros Express software. For each test, at least 30 nematodes were picked for assay. Three independent repeat experiments were performed. 2.9. Statistical analysis All data in this article were expressed as means7 SD. Graphs were generated using Microsoft Excel (Microsoft Corp., Redmond, WA). Analysis of variance (ANOVA) followed by a Dunnett’s t-test was used to determine the significance of the differences between the groups. The probability levels of 0.05 and 0.01 were considered statistically significant.
3. Results 3.1. Ca exposure at higher concentrations reduces the lifespan of wild-type N2 nematodes We first examined the effects of Ca exposure at different concentrations on the lifespan in adult wild-type N2 animals grown at 20 1C. As shown in Fig. 1, adult wild-type nematodes grown under our laboratory conditions had a mean lifespan of 17.5 days and an average maximum lifespan of 27.5 days at 20 1C. Exposure to 0.78 mM of Ca would not obviously affect the mean lifespan and maximum lifespan of treated wild-type nematodes. However, exposure to Ca at concentrations from 1.56 to 100 mM could obviously decrease the maximum lifespan of wild-type animals by 2.9%, 11.6%, 15.6%, 22.5%, 24.4%, 26.9% and 33.8%. Similarly, exposure to Ca at concentrations from 1.56 to 100 mM could significantly (1.56 mM, Po0.05; 3.13–100 mM, Po0.01) reduce the mean
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lifespan of wild-type animals by 12.6%, 15.4%, 17.7%, 21.1%, 31.4%, 38.9% and 50.9%. Moreover, we saw an obvious increase in the internal Ca levels in the exposed nematodes with the increased supplementation of Ca, suggesting that the Ca supplemented was taken up by the nematodes. Thus, Ca exposure at concentrations more than 1.56 mM will severely reduce lifespan of wild-type N2 nematodes. 3.2. The shortened lifespan in Ca exposed nematodes may be due to changed aging In order to validate the lifespan results, formation of lipofuscin was analyzed in Ca exposed nematodes. The lipofuscin is composed of cross-linked protein residues that irreversibly form due to oxidative processes (Brunk and Terman, 2002; Pluskota et al., 2009). To monitor the aging process, we examined the accumulation of intestinal autofluorescence in Ca exposed adult nematodes. As shown in Fig. 2, exposure to Ca at concentrations from 1.56 to 100 mM could induce a more rapid accumulation of intestinal autofluorescence than control at day 12 (Po0.01). Nematodes exposed to Ca at concentrations from 6.25 mM to 100 mM also displayed a remarkably increased lipofuscin-like intestinal autofluorescence at day 8 (6.25 mM, Po0.05; 12.5–100 mM, Po0.01). Moreover, early at day 4, nematodes exposed to Ca at concentrations from 25 mM to 100 mM could accumulate intestinal autofluorescence more rapidly than control (25 mM, Po0.05; 50–100 mM, Po0.01). These results were consistent with the lifespan analysis above, suggesting that the observed short-lived phenotype induced by Ca exposure may be due to the formation of age-related oxidative processes in C. elegans. 3.3. Ca exposure at higher concentrations induces the severe stress response Again, we explored the stable transgenic line of KC136 to investigate the possible stress response induced by Ca exposure at higher concentrations. Stress response was monitored by the significant induction of hsp-16.2::gfp expression (more than 50% of a population, above the line). As shown in Fig. 3, exposure to Ca at the concentration of 0.78 mM could not induce a severe stress response. In contrast to this, exposure to Ca at concentrations of 1.56 and 3.13 mM induced a moderate but significant (Po0.05) induction of hsp-16.2::gfp expression compared to control. Furthermore, nematodes exposed to Ca at concentrations more than 6.25 mM showed a very severe stress response as reflected by the noticeable (Po0.01) induction of hsp-16.2::gfp expression compared to control. This observation further suggests that Ca exposure at higher concentrations may induce the severe stress response in C. elegans. 3.4. Exposure to a high concentration of Ca with Cd induces the combinational toxicity on lifespan in wild-type N2 nematodes We next investigated the possible effects of combined Ca/Cd exposure on lifespan in wild-type N2 nematodes. As shown in Fig. 4, under our experimental conditions, exposure to 200 mM of Cd resulted in noticeably reduced maximum lifespan and mean lifespan compared to control. Combined exposure to Ca (25 mM)/ Cd (200 mM) could decrease the maximum lifespan by 15.9% compared to single exposure to Ca (25 mM), and by 17.5% compared to single exposure to Cd (200 mM), respectively; however, no difference could be found for maximum lifespan between combined exposure to Ca (1.56 mM)/Cd (200 mM) and single exposure to Cd (200 mM). Similarly, combined exposure to
Fig. 1. Effects of Ca exposure on lifespan in wild-type N2 nematodes. (A) Effects of Ca exposure at different concentrations on lifespan of nematodes grown at 20 1C. (B) Comparison of mean lifespans in nematodes exposed to Ca at different concentrations. (C) Internal Ca concentration determination using ICP-MS. Data are expressed as mean 7 SD. nPo 0.05 vs. 0 mM, nnP o0.01 vs. 0 mM.
Ca (25 mM)/Cd (200 mM) could reduce the mean lifespan by 40% compared to single exposure to Ca (25 mM), and by 25% compared to single exposure to Cd (200 mM), respectively. No differences could be observed for mean lifespan between combined exposure to Ca (1.56 mM)/Cd (200 mM) and single exposure to Cd (200 mM). These results demonstrate that the combined exposure to a high concentration (25 mM) of Ca with Cd will induce an obviously synergistic toxicity on lifespan in nematodes.
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Ca (1.56 mM)/Cd (200 mM) was similar to that in nematodes exposed to Cd (200 mM). Therefore, the exposure to high level of Ca may be required for inducing the obviously combined Ca/Cd toxicity on lifespan by activating more severe stress response in nematodes. 3.6. Combined exposure to a high concentration of Ca with Cd induces more severe oxidative stress than single metal exposure in wild-type N2 nematodes
Fig. 2. Effects of Ca exposure at different concentrations on mean autofluorescence from intestine lipofuscin in day 4, day 8 and day 12 adult nematodes. Data are expressed as mean 7SD. nPo 0.05 vs. 0 mM, nnP o 0.01 vs. 0 mM.
Fig. 3. Effects of Ca exposure on stress responses in wild-type N2 nematodes. Significant induction of hsp-16.2::gfp expression (more than 50% of a population, above the line) was observed in wild-type N2 nematodes exposed to Ca at concentrations more than 1.56 mM compared to control. Data are expressed as mean 7 SD. nP o 0.05 vs. 0 mM, nnPo 0.01 vs. 0 mM.
We further ask whether the induction of combined Ca/Cd toxicity on lifespan is enhancing the damage from oxidative stress. SOD belongs to the major defense enzymes against superoxide radicals, and its activities are directly linked to oxidative stress (Yanase et al., 2002; Wang and Wang, 2008b). As shown in Fig. 6A and B, under our experimental conditions, exposure to Ca at concentrations of 25 mM (Po0.01) and 1.56 mM (Po0.05), and to Cd at the concentration of 200 mM (Po 0.01) all significantly decreased the SOD activities in exposed nematodes compared to control. Combined exposure to Ca (25 mM)/Cd (200 mM) could cause a more severe reduction in SOD activity than single exposure to Ca (25 mM) or to Cd (200 mM), whereas the SOD activity in nematodes exposed to Ca (1.56 mM)/Cd (200 mM) was similar to that in nematodes exposed to Cd (200 mM). The observations above were further confirmed by the analysis of oxidative damage. As shown in Fig. 6C and D, exposure to Ca at concentrations of 25 (Po0.01) and 1.56 mM (P o0.05), and to Cd at the concentration of 200 mM (Po0.01) all significantly increased the oxidative damage in exposed nematodes compared to control. Furthermore, combined exposure to Ca (25 mM)/ Cd (200 mM) induced a more severe increase in oxidative damage in exposed nematodes than single exposure to Ca (25 mM) or to Cd (200 mM); however, no significant alterations of oxidative damage could be detected in nematodes exposed to Ca (1.56 mM)/ Cd (200 mM) from those in nematodes exposed to Cd (200 mM). Therefore, combined exposure to a high concentration (25 mM) of Ca with Cd may induce the synergistic toxicity on lifespan by activating more severe oxidative stress in nematodes. 3.7. Combinational Ca/Cd toxicity on lifespan is dependent of the expression of mev-1
To examine whether the effect of 1.56 mM Ca may be masked by the strong effect of Cd, we further selected an intermediate range of Ca (12.5 mM) to investigate the combined exposure to Ca (12.5 mM)/Cd (200 mM) on lifespan, and combined exposure to Ca (12.5 mM)/Cd (200 mM) could noticeably decrease the lifespan compared to single exposure to Ca (12.5 mM) or to Cd (200 mM). These data imply that Ca at 1.56 mM may still have effect with Cd at 200 mM, but the difference was not detectable in our assay.
3.5. Combined exposure to a high concentration of Ca with Cd activates more severe stress response than single metal exposure in wild-type N2 nematodes We next determined the possible effects of combined exposure to a high concentration of Ca with Cd on stress response. As shown in Fig. 5, exposure to 200 mM of Cd would cause a noticeable induction of hsp-16.2::gfp expression (more than 50% of a population, above the line) compared to control. Moreover, combined exposure to Ca (25 mM)/Cd (200 mM) resulted in a more significant induction of hsp-16.2::gfp expression than single exposure to Ca (25 mM) or to Cd (200 mM). Different from this, the induction of hsp-16.2::gfp expression in nematodes exposed to
The gene mev-1 encodes a subunit of the enzyme succinate dehydrogenase cytochrome b, which is a component of complex II of the mitochondrial electron transport chain (Ishii et al., 1998). mev-1 governs the rate of ageing by modulating the cellular response to oxidative stress in C. elegans (Ishii et al., 1998). The mev-1 mutant is significantly short-lived with several phenotypes consistent with elevated oxidative stress (Lin et al., 2006). Again, we investigated the effects of mev-1 mutation on the formation of combined Ca/Cd toxicity on lifespan in nematodes. As shown in Fig. 7, exposure to Ca (25 mM) or Cd (200 mM) more noticeably reduced the maximum and mean lifespans in mev-1 mutant nematodes than those in wild-type N2 nematodes. In addition, both the maximum lifespan and mean lifespan in Ca (25 mM)/ Cd (200 mM) exposed mev-1 mutant nematodes were shorter that those in Ca (25 mM)/Cd (200 mM) exposed wild-type N2 nematodes. Moreover, mutation of mev-1 could not block the formation of combinational Ca/Cd toxicity on lifespan. Nevertheless, mutation of mev-1 obviously enhanced the combined Ca/Cd toxicity on lifespan in nematodes. Under our experimental conditions, combined exposure to Ca (25 mM)/Cd (200 mM) could decrease the mean lifespan by approximately 2.4 days compared to single exposure to Cd (200 mM) in wild-type N2 nematodes. In contrast,
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Fig. 4. Effects of combined Ca/Cd exposure on lifespan in wild-type N2 nematodes. (A) Effects of combined exposure of Ca at the concentration of 25 mM with Cd (200 mM) on lifespan of nematodes grown at 20 1C. (B) Effects of combined exposure of Ca at the concentration of 1.56 mM with Cd (200 mM) on lifespan of nematodes grown at 20 1C. (C) Effects of combined exposure of Ca at the concentration of 12.5 mM with Cd (200 mM) on lifespan of nematodes grown at 20 1C. (D) Combined exposure with Ca (25 mM)/Cd (200 mM) induced a more decreased lifespan than single exposure to Ca (25 mM) or Cd (200 mM). (E) Combined exposure with Ca (1.56 mM)/Cd (200 mM) induced a similarly decreased lifespan to single exposure to Cd (200 mM). (F) Combined exposure with Ca (12.5 mM)/Cd (200 mM) induced a more decreased lifespan than single exposure to Cd (200 mM). Control, without metal exposure. NS, no significant difference. Data are expressed as mean 7 SD. nnPo 0.01.
combined exposure to Ca (25 mM)/Cd (200 mM) reduced the mean lifespan by approximately 4.1 days compared to single exposure to Cd (200 mM) in mev-1 mutant nematodes, suggesting that the formation of combinational Ca/Cd toxicity on lifespan is dependent on the expression of mev-1.
3.8. Insulin signaling pathway regulates the formation of synergistic Ca/Cd toxicity on lifespan in nematodes In C. elegans, the lifespan is regulated hormonally by an insulin/IGF-like signaling pathway. In DAF-2 pathway mutants,
DAF-16, a fork-head-family transcription factor, accumulates in the nuclei of many cell types, where it results in changes in the expression of a wide variety of response, and thereby extends lifespan (Braeckman and Vanfleteren, 2007). Previous study has demonstrated that mutation of daf-2 can result in increased metal resistance (Barsyte et al., 2001). To examine whether the insulin signaling pathway is involved in the regulation of synergistic Ca/Cd toxicity on lifespan, we next investigated the effects of daf-16 and daf-2 mutation on the formation of synergistic Ca/Cd toxicity on lifespan in nematodes. The mean lifespan in Ca (25 mM)/Cd (200 mM) exposed daf-16 mutant nematodes was shorter than those in Ca (25 mM)/Cd (200 mM) exposed
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Fig. 5. Effects of combined Ca/Cd exposure on stress responses in wild-type N2 nematodes. (A) Effects of combined exposure of Ca at the concentration of 25 mM with Cd (200 mM) on the induction of hsp-16.2::gfp expression. (B) Effects of combined exposure of Ca at the concentration of 1.56 mM with Cd (200 mM) on the induction of hsp-16.2::gfp expression. Stress response was monitored by the significant induction of hsp-16.2::gfp expression (more than 50% of a population, above the line). Control, without metal exposure. NS, no significant difference. Data are expressed as mean 7 SD. nnP o 0.01.
Fig. 6. Effects of combined Ca/Cd exposure on superoxide dismutase (SOD) activity, and oxidative damage in wild-type N2 nematodes. (A) Effects of combined exposure of Ca at the concentration of 25 mM with Cd (200 mM) on SOD activity. (B) Effects of combined exposure of Ca at the concentration of 1.56 mM with Cd (200 mM) on SOD activity. (C) Effects of combined exposure of Ca at the concentration of 25 mM with Cd (200 mM) on oxidative damage. (D) Effects of combined exposure of Ca at the concentration of 1.56 mM with Cd (200 mM) on oxidative damage. Control, without metal exposure. NS, no significant difference. Data are expressed as mean 7 SD. n Po 0.05 vs. control, nnPo 0.01 vs. control (if not specially indicated).
wild-type N2 nematodes (Fig. 8A). In contrast, the mean lifespan in Ca (25 mM)/Cd (200 mM) exposed daf-2 mutant nematodes was longer than those in Ca (25 mM)/Cd (200 mM) exposed wild-type N2 nematodes (Fig. 8B). In addition, mutation of daf-16 and daf-2 could not block the formation of synergistic Ca/Cd toxicity on nematode longevity (Fig. 8A and B). Nevertheless, mutation of daf-16 could obviously enhance the synergistic Ca/Cd toxicity on nematode longevity, but mutation of daf-2 could noticeably alleviate the synergistic Ca/Cd toxicity on nematode longevity.
Under our experimental conditions, combined exposure to Ca (25 mM)/Cd (200 mM) reduced the mean lifespan by approximately 3.6 days compared to single exposure to Cd (200 mM) in daf-16 mutant nematodes (Fig. 8A), and combined exposure to Ca (25 mM)/Cd (200 mM) reduced the mean lifespan by approximately 1.4 days compared to single exposure to Cd (200 mM) in daf-2 mutant nematodes (Fig. 8B). These data suggest that the formation of synergistic Ca/Cd toxicity on lifespan is at least partially dependent of the insulin signaling.
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In C. elegans, DAF-16 activities in the intestine and nervous system could completely or significantly restore the longevity of daf-16(-) germline-deficient nematodes, and increase the lifespans of daf-16(-) insulin/IGF-1-pathway mutants substantially (Libina et al., 2003). We further examined the tissue-specific activities of DAF-16 in the regulation of the synergistic Ca/Cd toxicity on lifespan of nematodes. The effects of Ca (25 mM)/ Cd (200 mM) exposure on the lifespan of daf-16;daf-2 mutant nematodes were similar to those on the lifespan of wild-type N2 nematodes (data not shown). The mean lifespan in Ca (25 mM)/ Cd (200 mM) exposed daf-16;daf-2;muEx211/Pges-1::gfp::daf-16 and daf-16;daf-2;muEx169/Punc-119::gfp::daf-16 nematodes was longer than those in Ca (25 mM)/Cd (200 mM) exposed wild-type N2 nematodes (Fig. 8C and D). Similarly, over-expression of intestinal DAF-16 (Pges-1::gfp::daf-16) and neuronal DAF-16 (Punc-119::gfp::daf-16) could not block the formation of synergistic Ca/Cd toxicity on lifespan of daf-16;daf-2 mutant nematodes (Fig. 8C and D). Nevertheless, over-expression of intestinal and neuronal DAF-16 could significantly alleviate the synergistic Ca/Cd toxicity on lifespan of nematodes. Combined exposure to Ca (25 mM)/Cd (200 mM) reduced the mean lifespan by approximately 1.1 days compared to single exposure to Cd (200 mM) in
Fig. 7. Effects of combined Ca/Cd exposure on lifespan of mev-1 nematode mutant. (A) Effects of combined exposure of Ca at the concentration of 25 mM with Cd (200 mM) on lifespan in wild-type N2 and mev-1 mutant nematodes grown at 20 1C. (B) Mean lifespan comparison in wild-type N2 and mev-1 mutant nematodes. Data are expressed as mean 7 SD. nPo 0.05 vs. mev-1, nnP o 0.01 vs. N2 or mev-1 (if not specially indicated).
Fig. 8. Effects of insulin signaling on the formation of synergistic Ca/Cd toxicity on lifespan in nematodes. (A) Effects of combined exposure of Ca at the concentration of 25 mM with Cd (200 mM) on mean lifespan in wild-type N2 and daf-16 mutant nematodes grown at 20 1C. (B) Effects of combined exposure of Ca at the concentration of 25 mM with Cd (200 mM) on mean lifespan in wild-type N2 and daf-2 mutant nematodes grown at 20 1C. (C) Effects of combined exposure of Ca at the concentration of 25 mM with Cd (200 mM) on mean lifespan in wild-type N2 and daf-16;daf-2;muEx211 mutant nematodes grown at 20 1C. (D) Effects of combined exposure of Ca at the concentration of 25 mM with Cd (200 mM) on mean lifespan in wild-type N2 and daf-16;daf-2;muEx169 mutant nematodes grown at 20 1C. Data are expressed as mean 7SD. nP o 0.05 vs. wild-type or mutant (if not specially indicated), nnP o0.01 vs. wild-type or mutant (if not specially indicated).
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daf-16;daf-2;muEx211 mutant nematodes (Fig. 8C), and combined exposure to Ca (25 mM)/Cd (200 mM) reduced the mean lifespan by approximately 1.8 days compared to single exposure to Cd
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Fig. 9. Effects of metal exposure on brood size (A) and body size (B). Data are expressed as mean 7 SD. nP o 0.05 vs. control,
(200 mM) in daf-16;daf-2;muEx169 mutant nematodes (Fig. 8D). Therefore, the intestinal and neuronal DAF-16 activities are involved in the control of synergistic Ca/Cd toxicity lifespan of nematodes. 3.9. Effects of Ca and combined Ca/Cd exposure on brood size and nematode development We finally investigated the effects of Ca and combined Ca/Cd exposure on brood size and development in nematodes. As shown in Fig. 9, exposure to Ca at concentrations from 0.78 to 25 mM could not cause obvious decrease in brood size and body size, whereas the noticeable decrease in brood size or body size could be observed in nematodes exposed to 50 and 100 mM of Ca. In addition, exposure to Cd (200 mM) and combined Ca (25 mM)/ Cd (200 mM) could also result in significant decreases in brood size and body size compared to control. Moreover, no obvious differences could be found for brood size and body size in nematodes exposed to Cd (200 mM) from those in nematodes exposed to Ca (25 mM)/Cd (200 mM). Furthermore, exposure to Ca at different concentrations and combined Ca (25 mM)/ Cd (200 mM) could not induce severe deficits in dauer formation compared to control (data not shown).
4. Discussion In the present study, we first provide the evidence to indicate the important roles of Ca exposure at different concentrations in influencing the lifespan of nematodes. Ca exposure at concentrations more than 1.56 mM can induce the severely reduced lifespan in wild-type N2 nematodes (Fig. 1). In addition, the analysis on the accumulation of intestinal autofluorescence suggested that the observed short-lived nematodes appeared after Ca exposure may be due to accelerated aging, and not due to an unrelated, pleiotropic cause (Fig. 2). These conclusions on the Ca toxicity on lifespan in nematodes are largely consistent with the observations in other animals and even in plants. The average lifespan of the rotifer Asplanchna brightwelli could be significantly reduced when 1.2 or 2.4 mM CaCl2 was added to the culture medium, and the maximum lifespan was also reduced as the CaCl2 concentration was increased (Enesco and Holtzman, 1980). Studies on the function of PPF1 during plant development further suggest that internal Ca2 + concentration or calcium storage capacity is closely related to the induction of cell death or cell senescence in plant cells (Li et al., 2004; Wang et al., 2008a).
P o 0.01 vs. control.
nn
The contribution of the release of calcium from smooth endoplasmic reticulum calcium stores can also be altered with age in peripheral neurons (Buchholz et al., 2007). In addition, the exposure of hippocampal neurons to Ab peptides led to dendritic dystrophy and activation of apoptotic neuronal death that may be associated with a significant rise in cytosolic Ca2 + concentrations (Resende et al., 2007). In contrast, inhibition of Ca2 + influx during arsenic treatment reduced the cell death in Leishnania spp. (Mehta and Shaha, 2006). Similarly, simultaneous treatment with either verapamil (an inhibitor of Ca2 + through plasma membrane) or dantrolene (an inhibitor of mobilization of [Ca2 + ]i from endoplasmic reticulum) or BAPTA (a Ca2 + chelator) could obviously reduce Ni compound-induced DNA single-strand breaks in both chromosomal and nuclear chromatin (M’Bemba-Meka et al., 2005). Previous study has demonstrated that the synthetic SOD/CAT mimetics (SCMs), Euk-134 and Euk-8 confer resistance to the oxidative stress-inducing agent, paraquat and to thermal stress, and the lifespan of nematodes can be extended by the administration of Euk-134 and Euk-8 without any toxic effects on development and fertility, suggesting the involvement of oxidative stress in regulating nematode lifespan (Sampayo et al., 2003). The cellular Ca2 + levels hold the key to activating many response pathways, and high [Ca2 + ]i can cause disruption of mitochondrial Ca2 + equilibrium, which will result in reactive oxygen species (ROS) formation due to the stimulation of electron flux along the electron transport chain (Chacon and Acosta, 1991). In the current work, our data further suggest that Ca exposure at higher concentrations results in the severe stress response as monitored by the significantly elevated induction of hsp-16.2::gfp expression (Fig. 3). Moreover, the severe oxidative stress could be induced by the Ca exposure at higher concentrations as revealed by the decreased SOD activities, and the oxidative damage in C. elegans (Fig. 6). It was also reported that exposure to metals, such as Cu and Ba, may usually stimulate the formation of oxidative stress in animal cells (Nawaz et al., 2006; Wang and Wang, 2008b). Our data in this project further indicate the combined Ca/Cd toxicity on lifespan in C. elegans. Combined exposure to Ca (25 mM)/Cd (200 mM) could significantly decrease the maximum and mean lifespans compared to single exposure to Cd (200 mM); however, no difference could be found for maximum lifespan, and mean lifespan between combined exposure to Ca (1.56 mM)/Cd (200 mM) and single exposure to Cd (200 mM) (Fig. 4), suggesting that combined exposure to a high concentration of Ca with Cd can induce the combined toxicity on lifespan in wild-type N2 nematodes. Similarly, the combined Ca/Cd toxicity on lifespan
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could be observed in exposed planarians (Wang et al., personal communication). It was also reported that the copper toxicity increased at higher Ca:Mg ratios for Daphnia magna (Naddy et al., 2002). The main reason to select the Cd for the assay of combined toxicity on lifespan is that the toxicology from Cd exposure is the most well studied so far in C. elegans (Swain et al., 2004). At least four lines of reasoning made us select the Ca (25 mM) to perform the study on combinational Ca/Cd toxicity on nematode lifespan. First, exposure to 25 mM of Ca obviously reduced the maximum and mean lifespans of wild-type N2 nematodes (Fig. 1). Second, exposure to 25 mM of Ca could cause severe stress response (Fig. 3) and oxidative stress (Fig. 6). Third, exposure to 25 mM of Ca did not obviously alter the nematodes’ dauer formation (data not shown), brood size and body size (Fig. 9). Fourth, Ca (25 mM) exposure would not influence the dauer formation (data not shown), brood size and body size (Fig. 9) of nematodes exposed to Cd (200 mM). For the regulation mechanisms explaining the formation of combined Ca/Cd toxicity on nematode lifespan, we raised the notion that one of the mechanisms is the possible involvement of oxidative stress in the regulation of combined Ca/Cd toxicity on nematode lifespan. Combined exposure to Ca (25 mM)/ Cd (200 mM) could result in a more significant induction of hsp-16.2::gfp expression, a more severe reduction of SOD activity, and a more severe increase in oxidative damage than single exposure to Cd (200 mM) (Figs. 5 and 6), suggesting that combined exposure to a high concentration (25 mM) of Ca with Cd may cause the synergistic toxicity on lifespan by inducing more severe oxidative stress in nematodes. Moreover, because mutation of mev-1 could obviously enhance the synergistic Ca/Cd toxicity on nematode lifespan, the formation of combined Ca/Cd toxicity on lifespan is dependent of the expression of mev-1 (Fig. 7). Nevertheless, mutation of mev-1 could not block the formation of combined Ca/Cd toxicity on lifespan (Fig. 7). Similarly, induction of oxidative stress at early L2-larval stage by paraquat (2 mM) treatment could not also block the formation of combined Ca/Cd toxicity on lifespan (data not shown). These observations suggest that occurrence of severe oxidative stress may still not be the only mechanism to explain the formation of combined Ca/Cd toxicity on nematode lifespan. Another possible mechanism we raised to explain the formation of combined Ca/Cd toxicity on lifespan is the involvement of insulin signaling pathway. In the present study, our data suggest that the formation of combined Ca/Cd toxicity on lifespan is at least partially dependent on the insulin signaling, since mutation of daf-16 obviously enhanced the combined Ca/Cd toxicity on lifespan and mutation of daf-2 noticeably alleviated the combined Ca/Cd toxicity on lifespan (Fig. 8). Moreover, because overexpression of intestinal and neuronal DAF-16 could significantly alleviate the combined Ca/Cd toxicity on lifespan of daf-16;daf-2 mutant nematodes (Fig. 8), and over-expression of muscle DAF-16 had no noticeable effects on the formation of combined Ca/Cd toxicity on lifespan of daf-16;daf-2 mutant nematodes (data not shown), the intestinal and neuronal DAF-16 activities may be involved in the control of combined Ca/Cd toxicity on lifespan in C. elegans. Nevertheless, mutation of daf-16 and daf-2, as well as the over-expression of intestinal and neuronal DAF-16, could not block the formation of combined Ca/Cd toxicity on lifespan, which implies that multiple signaling pathways may regulate the formation of combined Ca/Cd toxicity on lifespan of nematodes. Because both the exposure to Ca (25 mM) and the exposure to Ca (25 mM)/Cd (200 mM) could not induce severe deficits in dauer formation compared to control (data not shown), so at least two signaling pathways, oxidative stress and insulin signaling pathway, may play important roles in regulating the formation of combined Ca/Cd toxicity on lifespan in C. elegans.
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5. Conclusions In conclusion, Ca exposure at concentrations more than 1.56 mM induces the severely reduced lifespan in wild-type N2 nematodes. Combined exposure to Ca (25 mM)/Cd (200 mM) significantly decreases the lifespans compared to single exposure to Cd (200 mM). Moreover, combined exposure to Ca (25 mM)/ Cd (200 mM) results in a more significant induction of hsp16.2::gfp expression, a more severe reduction in SOD activity, and a more severe increase in oxidative damage than single exposure to Cd (200 mM). Furthermore, both the oxidative stress and the insulin signaling pathway are involved in regulating the formation of combined Ca/Cd toxicity on lifespan in C. elegans.
Acknowledgments Strains used in this study were provided by the Caenorhabditis Genetics Center (Funded by the NIH, National Center for Foundation from Research Resource, USA). This work was supported by the National Natural Science Foundation of China (nos. 30771113 and 30870810).
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