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Exposure to metals induces morphological and functional alteration of AFD neurons in nematode Caenorhabditis elegans
Environmental Toxicology and Pharmacology 28 (2009) 104–110
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Exposure to metals induces morphological and functional alteration of AFD neurons in nematode Caenorhabditis elegans Xiaojuan Xing, Min Du, Xuemei Xu, Qi Rui, Dayong Wang ∗ Key Laboratory of Developmental Genes and Human Disease in Ministry of Education, Department of Genetics and Developmental Biology, Southeast University Medical School, Nanjing 210009, China
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Article history: Received 5 December 2008 Received in revised form 4 March 2009 Accepted 4 March 2009 Available online 17 March 2009 Keywords: Metal exposure Thermotaxis AFD neuron ttx-1 Caenorhabditis elegans
a b s t r a c t Previous studies have revealed that metal exposure will cause severe deficits in perception behaviors. Here we investigated the effects of metal (Hg, Cu, Ag, and Cr) exposure on thermotaxis to cultivation temperature in Caenorhabditis elegans. Our data suggest that exposure to higher concentrations of examined metals induced severe deficits in thermotaxis, and a significant reduction in thermotaxis could be even observed in nematodes exposed to 2.5 M of Hg. Moreover, exposure to higher concentrations of examined metals and 2.5 M of Hg induced significant decreases in relative intensities and relative sizes of fluorescent puncta of cell bodies in AFD thermosensory neurons. In addition, exposure to higher concentrations of examined metals resulted in a significant reduction in relative intensities and relative lengths of sensory endings in AFD neurons. Furthermore, the relative transcript levels of ttx-1, which functions in specifying the fate of AFD neuron, were significantly decreased in nematodes exposed to 2.5 M of Hg, and 50 and 100 M of examined metals. Thus, metal exposure at high concentrations will induce the severe deficits in thermotaxis to cultivation temperature possibly by altering the morphology or development of AFD neuron and damaging the molecular basis for function of AFD neuron in nematodes. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Nematode Caenorhabditis elegans, a model animal best characterized at the genetic, physiological, molecular, and developmental levels (Riddle et al., 1997), has already been explored as a valuable bioindicator (Williams and Dusenbery, 1990a; Donkin and Williams, 1995). In addition, it has been proved that C. elegans can serve as an excellent candidate for studying the neurotoxicology, since it has only 302 neurons and the complete writing diagram for chemical and electrical connections is available (White et al., 1986; Leung et al., 2008). C. elegans can be used to test the environmental contaminants known to have neural toxic effects. The deficits in movement behavior can be monitored using a computer tracking system after exposure to metals or organophosphorus insecticides in nematodes (Dhawan et al., 2000; Anderson et al., 2001; Boyd et al., 2003; Anderson et al., 2004; Rajini et al., 2008). The toxic effects of metal exposure on endpoints of head thrash, body bend, and basic movements (forward sinusoidal movement (forward turn), reversal movement (backward turn), and turn) have been examined in nematodes (Wang et al., 2007a,b; Wang and Xing, 2008; Wang and Wang, 2008a,b). C. elegans was also used for the test of organophosphate-induced neurotoxicity by anticholinesterase
∗ Corresponding author. Tel.: +86 25 83272314 817; fax: +86 25 83324887. E-mail address: [email protected] (D. Wang). 1382-6689/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2009.03.006
activity assay (Cole et al., 2004; Rajini et al., 2008). Furthermore, exposure to metals of Al or Pb at high concentrations could induce severe decrease in memory behaviors (Ye et al., 2008a,b). Previous studies have revealed that metal exposure at higher concentrations will result in severe deficits in color vision, hearing ability, smelling ability, and olfactory function (Rose et al., 1992; Sulkowski et al., 2000; Wu et al., 2000; Urban et al., 2003; Gobba and Cavalleri, 2003). Thermotaxis is based on the thermal sense, which is an important perception behavior for the life of the animals (Mori and Ohshima, 1997). Temperature is a critical modulator of metabolism and behavior, and sensed by C. elegans using the AFD thermosensory neurons (Satterlee et al., 2001). In addition, nematodes grown in the presence of food at a particular temperature will actively seek this temperature when presented with a thermal gradient but will avoid this temperature if it is associated with an absence of food (Hedgecock and Russell, 1975; Mori, 1999). ttx-1, a homolog of otd/Otx, specifies the thermotaxis neuron fate in C. elegans, and misexpression of ttx-1 can convert other sensory neurons to an AFD-like fate (Satterlee et al., 2001). Thus, one of the objectives of this study was to investigate the possible effects of metal exposure on the thermotaxis to cultivation temperature in C. elegans. Four metals were selected, and they were Cr, Cu, Hg, and Ag, respectively. In addition, our specific aims were to examine the effects of metal exposure on the structures or morphologies of AFD sensory neurons and the expression patterns of ttx-1 gene in nematodes. The morphological changes of cell body
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and sensory ending of AFD thermosensory neurons and expression patterns of ttx-1 gene were examined in control and metal exposed nematodes.
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of ttx-1 was expressed as the ratio of ttx-1/act-1. At lease three replicates were performed for statistical purposes. 2.5. Statistical analysis
2. Materials and methods 2.1. Reagents and strains The metal concentrations used in this study were selected as previously described (Wang and Yang, 2007). Four concentrations of CrCl2 , CuSO4 , HgCl2, and AgNO3 solutions were used in the current work, and they were 2.5, 50, 100 and 150 M, respectively. Metal concentrations of exposed solutions were analyzed by atomic absorption spectrophotometry (AAS; Pye-Unicam model SP9, Cambridge, UK). All the chemicals were obtained from Sigma–Aldrich (St. Louis, MO, USA). Nematodes used in the present study were wild-type N2 and DA1267 (lin15(n765); dEx1267[lin-15(+) gcy-8::GFP]) labeling the AFD thermosensory neuron, originally obtained from the Caenorhabditis Genetics Center (funded by the NIH National Center for Research Resource, USA). They were maintained on nematode growth medium (NGM) plates seeded with Escherichia coli OP50 at 20 ◦ C as described (Brenner, 1974). Gravid nematodes were washed off the plates into centrifuge tubes, and were lysed with a bleaching mixture (0.45N NaOH, 2% HOCl). Age synchronous populations of larva (L4-stage) were obtained by the collection as described (Donkin and Williams, 1995). The L4-larva stage nematodes were washed with doubledistilled 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, 1990b). Exposures were performed in 12-well sterile tissue culture plates as described (Mutwakil et al., 1997). All exposures were 24-h long and were carried out in 20 ◦ C incubator in the presence of food. 2.2. Thermotaxis assay The method was performed as described (Gomez et al., 2001; Ye et al., 2008a). Approximately 50 nematodes were grown at 20 ◦ C overnight in the presence of a fresh lawn of the bacteria strain of OP50 on a 6 cm Petri dish. Young adults were transferred on to a fresh plate devoid of bacteria for 2 min, and individual nematodes were then deposited on a 9 cm Petri dish. A radial gradient of temperature was created by placing a vial containing frozen acetic acid on the bottom of the plate and incubating at 25 ◦ C for 90 min in the presence of a constant humidity of 60%. Upon removal of the nematodes from the plate, tracks left were photographed and analyzed. A trace is considered as isothermal if more than half of the trace length left on the agar surface by a single nematode is circular or presents an arc of circle near the isotherm of the cultivation temperature. Each data point represents 10 independent assays. 2.3. Fluorescence quantification To quantify fluorescence intensities, fluorescence images of neurons in control and metal exposed young adult nematodes were captured with a Zeiss Axiocam MRm camera on a Zeiss Axioplan 2 Imaging System with ×40 objective using SlideBook software (Intelligent Imaging Innovations). Images were acquired with a Quantix cooled CCD camera, and illumination was provided by a 175 W xenon arc lamp and Green Fluorescent Protein (GFP) filter sets. Exposure times were chosen to fill the 12-bit dynamic range without saturation, and out-of-focus light was removed with a constrained iterative deconvolution algorithm. Background fluorescence from the coverslip and from nonspecific tissue autofluorescence was removed by subtracting an image filtered with a low pass Gaussian filter. The puncta sizes for cell bodies or relative lengths of sensory endings were measured as the maximum radius for assayed fluorescent puncta. The relative fluorescence intensity of particular fusion proteins at the cell bodies or sensory endings was obtained by integrating pixel intensity in at least 20 nematodes. Similarly, the relative sizes of fluorescent puncta for cell bodies or relative lengths of sensory endings were also examined in at least 20 nematodes. The images were photographed and examined on the same day to avoid effects of light source variance on fluorescence intensity. 2.4. Semi-quantitative Reverse Transcriptase (RT)–Polymerase Chain Reaction (PCR) assay Total RNA was isolated using guanidinium thiocyanate/phenol method as described (Morse and Bass, 1999). Control and metal exposed nematodes were sampled for the RT–PCR assay. The frozen nematode pellets were harvested from 1 L of mixed stage liquid cultures. Purified Poly (A)+ RNA through two rounds of selection on oligo(dT)-cellulose was used for the RNA blotting. Genespecific primers were designed for ttx-1 (ttx-1F, 5 -ATGTCCTTGACGTCTTCC-3 , and ttx-1R, 5 -TTCCGTCCAAAGTGTAGG-3 ). Act-1 was used to determine the equal loading for each sample, and primers specific for act-1 were used in control reactions (act-1F, 5 -CGAAGCTTACCGTCCCAATCTACGAAG-3 ; act-1R, 5 TGAGAATTCGAAGCACTTGCGGTGAAC-3 ). Amplification of all DNA fragments for RT–PCR was performed for 30 cycles in a Perkin-Elmer 480 thermal cycler using a 55 ◦ C annealing temperature and a 1-min extension. The relative transcript level
All data in this article were expressed as means ± SD. Graphs were generated using Microsoft Excel (Microsoft Corp., Redmond, WA). One-way 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. Effects of metal exposure at different concentrations on the thermotaxis to cultivation temperature in C. elegans We first investigated the thermotaxis behaviors in nematodes exposed to Hg, Cu, Ag, and Cr at concentrations from 2.5 to 150 M. As shown in Fig. 1, exposure to 100 M (Cu, P < 0.05; Hg, Ag, and Cr, P < 0.01) and 150 M (P < 0.01) of all examined metals caused the significant decrease in percentage of nematodes performing isothermal tracking (IT) behavior after overnight feeding at 20 ◦ C. Exposure to 50 M of Hg (P < 0.01) and Cr (P < 0.05) could also result in the severe defects of thermotaxis to cultivation temperature, whereas exposure to 50 M of Ag and Cu did not alter the thermotaxis to cultivation temperature of exposed nematodes. Moreover, a significant (P < 0.05) reduction in thermotaxis to cultivation temperature could even be observed in nematodes exposed to 2.5 M of Hg. Therefore, exposure to higher concentrations of metals will induce severe deficits in thermotaxis to cultivation temperature, and the exposure to Hg can induce the most severe neurotoxicity on thermotaxis among the examined metals in C. elegans. 3.2. Effects of metal exposure at different concentrations on the cell body morphology of AFD sensory neuron in C. elegans Thermotaxis to cultivation temperature was under the control of AFD sensory neuron (Satterlee et al., 2001). Pgcy-8::GFP is a specific fluorescent marker to label the AFD sensory neuron (Satterlee et al., 2001). As shown in Fig. 2A and B, metal exposure could significantly change the relative fluorescent intensities of cell bodies in AFD neurons. Exposure to 100 M (Cu, P < 0.05; Hg, Ag, and Cr, P < 0.01) and 150 M (P < 0.01) of all examined metals caused the significant decrease in relative intensities of cell bodies in AFD neurons compared to control. Exposure to 50 M of Hg (P < 0.01) and Cr (P < 0.05) also induced a severe reduction in relative intensities of cell bodies in AFD neurons, whereas no remarkable alterations of relative intensities of cell bodies in AFD neurons could be observed in nematodes exposed to 50 M of Cu and Ag. In addition, a significant (P < 0.01) decrease in relative intensities of cell bodies in AFD neurons was further found in nematodes exposed to 2.5 M of Hg. No significant differences of relative intensities for cell bodies could be detected between AFDL and AFDR neurons in control and metal exposed nematodes. We also examined the effects of metal exposure at different concentrations on relative sizes of fluorescent puncta of cell bodies in AFD neurons. As shown in Fig. 2A and C, exposure to 100 and 150 M of all examined metals caused significant (P < 0.01) decrease in relative sizes of fluorescent puncta of cell bodies in AFD neurons compared to control. Moreover, exposure to 50 M of Hg, Ag, and Cr further induced the significant (P < 0.01) reduction of relative sizes of cell body fluorescent puncta in AFD neurons. In addition, exposure to 2.5 M of Hg also resulted in a significant (P < 0.01) decrease in relative sizes of cell body fluorescent puncta in AFD neurons. Similarly, no significant differences of relative sizes of cell body fluorescent puncta could be detected between AFDL and AFDR neurons in control and metal exposed nematodes. There-
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Fig. 1. . Effects of metal exposure at different concentrations on thermotaxis in C. elegans. Metals of Hg, Cu, Ag, and Cr were selected for the assay. Thermotaxis was evaluated by the percentage of nematodes performing isothermal tracking (IT) behavior after overnight feeding at 20 ◦ C. A trace is considered as isothermal if more than half of the trace length left on the agar surface by a single nematode is circular or present an arc of circle near the isotherm of the growth temperature. Bars represent means ± S.D. *P < 0.05 vs. 0 M; **P < 0.01 vs. 0 M.
fore, the morphology of cell bodies in AFD neurons can be severely altered by metal exposure to different degrees. 3.3. Effects of metal exposure at different concentrations on the sensory ending of AFD neuron in C. elegans Sensory cilia provide a compartment for the localization and exposure of signal transduction proteins to incoming sensory signals (Swoboda et al., 2000). AFD is a thermosensory neuron with a cilium exposed to the environment, and the finger-like microvilli can be observed at the end of the AFD dendrites (Swoboda et al., 2000). We next investigated the effects of metal exposure on the relative fluorescent intensities of sensory endings in AFDL neurons (Fig. 3A and B). The sensory endings of the AFD neurons are typically elaborate, consisting of a single cilium and numerous microvillar “fingers” (Fig. 3A). We found that, very different from the effects on morphology of cell body, exposure to metals of Hg, Cu, Ag, and Cr at concentrations from 50 to 150 M all caused a significant (P < 0.01) reduction of relative fluorescent intensities of sensory endings in AFDL neurons compared to control. Moreover, the severe (P < 0.01) decrease in relative fluorescent intensity of sensory ending in AFDL neuron was also detected in nematodes exposed to 2.5 M of Hg. In addition, no significant differences of relative fluorescent intensities of sensory endings could be detected between AFDL and AFDR neurons in control and metal exposed nematodes (data not shown). We further examined the effects of metal exposure on the relative lengths of sensory endings of AFDL neurons. As shown in Fig. 3A and C, exposure to 100 and 150 M of all examined metals resulted in the severe (P < 0.01) decrease in relative lengths of sensory endings in AFDL neurons compared to control. Furthermore, a significant reduction in relative lengths of sensory endings of AFDL neurons could be observed in nematodes exposed to 50 M of Hg (P < 0.01) and Cr (P < 0.05). In contrast, no significant alteration of relative lengths of sensory endings in AFDL neurons could be detected in nematodes exposed to 50 M of Cu and Ag, as well as to 2.5 M of all examined metals, compared to control. Similarly, no significant differences of relative lengths of sensory endings could be detected between AFDL and AFDR neurons in control and metal exposed nematodes (data not shown). In addition, no obvious
defects of dendrites and axon guidance were observed in nematodes exposed to all examined metals (data not shown). Therefore, metal exposure will alter the morphology and development or suppress the extension or growth of sensory ending of AFD cilium. 3.4. Effects of metal exposure at different concentrations on the expression levels of ttx-1 gene in C. elegans ttx-1 regulates all differentiated characteristics of the AFD neurons, and mutations of ttx-1 affect AFD neuron functions (Satterlee et al., 2001). We finally examined the changes of transcript levels of ttx-1 in control and metal exposed nematodes as revealed by RT–PCR assay. Because exposure to a very high concentration (150 M) of examined metals would severely affect the locomotion behaviors of exposed nematodes (data not shown), we only investigated the expression patterns of ttx-1 in control and metal exposed nematodes at concentrations not more than 100 M. As shown in Fig. 4, exposure to 2.5 M of Hg and Cr could induce the significant (P < 0.01) decrease in relative transcript levels of ttx-1 compared to control, but exposure to Cu and Ag at the same concentration would not affect the transcript levels of ttx-1 in exposed animals. Moreover, the significant decrease in relative transcript levels of ttx-1 could also be observed in nematodes exposed to all examined metals at concentrations of both 50 M (Cu, P < 0.05; Hg, Ag, and Cr, P < 0.01) and 100 M (P < 0.01) compared to control. The Northern blotting results were consistent with the observations by RT–PCR assay (data not shown). Therefore, metal exposure may result in the damage on the molecular basis for AFD neuron function to different degrees. 4. Discussion Perception to environmental cues is an important behavior for animals and human beings. Previous studies have revealed the possible effects of metal exposure on perception behaviors in animals. For example, exposure to mercury vapor can induce sub-clinical color vision impairment (Urban et al., 2003; Gobba and Cavalleri, 2003). A significant correlation between a high, long-term lead exposure index (defined by duration of employment and ambient
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Fig. 2. . Effects of metal exposure at different concentrations on morphology of AFD sensory neuron in C. elegans. Metals of Hg, Cu, Ag, and Cr were selected for the assay. (A) Morphological patterns of AFD sensory neurons. (B) Quantification of relative fluorescent intensities in cell bodies of AFD sensory neurons. (C) Relative sizes of fluorescent puncta for cell bodies of AFD sensory neuron. Pgcy-8::GFP labels the AFD sensory neurons. “L” indicates the cell body of left neuron; “R” indicates the cell body of right neuron. Bars represent means ± S.D. *P < 0.05 vs. 0 M; **P < 0.01 vs. 0 M.
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Fig. 3. . Effects of metal exposure on sensory ending (cilium) of AFD sensory neuron in C. elegans. Metals of Hg, Cu, Ag, and Cr were selected for the assay. (A) Morphological patterns of AFDL (the left one of paired AFD neurons) sensory neurons in nematodes exposed to metals. (B) Quantification of relative lengths of sensory endings of AFDL sensory neurons. (C) Relative sizes of fluorescent puncta for sensory endings of AFDL sensory neurons. Pgcy-8::GFP labels the AFD sensory neuron. Con, control without metal exposure. Bars represent means ± S.D. *P < 0.05 vs. 0 M; **P < 0.01 vs. 0 M.
lead concentration) and decreased hearing ability was found in two lead-battery manufacturing factories, suggesting that lead exposure might precipitate a more severe auditory than noise-exposure effect (Wu et al., 2000). Smell impairment caused by occupationally exposure to cadmium was also observed in workers (Sulkowski et al., 2000). Chronic occupational cadmium exposure sufficient to
cause renal damage is also associated with impairment in olfactory function (Rose et al., 1992). Although no changes in olfactory function as measured by either absolute threshold or two-odor discrimination tasks, nickel exposure could result in selective lesions to the olfactory system and decreases in the number of bipolar sensory receptor cells, the thickness of olfactory epithelium, and
Fig. 4. . Expression pattern changes of ttx-1 induced by metal exposure in C. elegans. Metals of Hg, Cu, Ag, and Cr were selected for the assay. (A) Semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction (RT–PCR) assay of expression patterns of ttx-1 gene. Act-1 was used to determine the equal loading for each sample. (B) Changes of relative transcript levels of ttx-1 in control and metal exposed nematodes. Bars represent means ± S.D. *P < 0.05 vs. 0 M; **P < 0.01 vs. 0 M.
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carnosine levels in rats (Evans et al., 1995). Multiple linear regression analysis relating to lead workers’ test performance and their lead exposure showed that performance on the sensory store memory test was significantly related to lead exposure (Williamson and Teo, 1986). Nevertheless, seldom studies have been performed to analyze the effects of metal exposure on the thermotaxis behaviors. In the present study, our data suggest that exposure to higher concentrations of our examined metals would induce severe deficits in thermotaxis to cultivation temperature, and the exposure to Hg could induce the most severe neurotoxicity on thermotaxis among the examined metals (Fig. 1). Exposure to higher concentrations of other heavy metals, such as Pb and Mn, also induced the severe neurotoxicity on thermotaxis in nematodes (D. Wang, personal communication). These observations further support the notion that exposure to high levels of toxic heavy metals may induce severe deficits in perception behaviors in animals. AFD sensory neuron plays a pivotal role in regulating the thermotaxis to cultivation temperature in nematodes (Mori, 1999; Satterlee et al., 2001). Our data suggest that morphologies or development of both the cell body and the sensory ending of AFD neurons can be severely influenced by metal exposure at relatively higher concentrations. Exposure to 100 and 150 M of all examined metals caused the significant decrease in relative intensities and relative sizes of fluorescent puncta of cell bodies in AFD neurons compared to control (Fig. 2). Similarly, exposure to 100 and 150 M of all examined metals caused the significant decrease in relative intensities and relative lengths of sensory endings in AFD neurons compared to control (Fig. 3). Moreover, the severe defects of relative intensities and relative sizes of fluorescent puncta of cell bodies, as well as relative intensities and relative lengths of sensory endings, in AFD neurons could be further detected in nematodes exposed to 50 M of Hg and Cr (Figs. 2 and 3). Thus, the morphology of AFD thermosensory neurons will be severely disrupted and the development of AFD thermosensory neurons will be noticeably inhibited by high level of metal exposure. Considering the fact that no obvious defects of dendrites and axon guidance could be observed in nematodes exposed to all examined metals (data not shown), the mainly altered structures by metal exposure may be cell body and sensory ending in AFD neurons. In addition, no significant differences of relative lengths of sensory endings could be detected between AFDL and AFDR neurons in control and metal exposed nematodes (data not shown). Nevertheless, no phenotype of loss of cilium as previously observed in daf-19 mutants could be found in nematodes exposed to all examined metals (Swoboda et al., 2000), suggesting that metal exposure will not influence the cilium formation process. Furthermore, the significant decrease in relative intensities or relative sizes of fluorescent puncta of cell bodies in AFD neurons could be further observed in nematodes exposed to 2.5 M of Hg. The severe decrease in relative fluorescent intensities of sensory endings in AFDL neurons could also be detected in nematodes exposed to 2.5 M of Hg. Together with the effects from exposure to 2.5 M of Hg on thermotaxis to cultivation temperature, our results demonstrated that Hg exposure can activate the most severe neurotoxicity on thermotaxis to cultivation temperature among the examined metals. ttx-1 functions is used in specifying the thermotaxis neuron fate, and is expressed in the AFD neurons (Satterlee et al., 2001). The significant decrease in relative transcript levels of ttx-1 could be detected in nematodes exposed to 2.5 M of Hg, and 50 and 100 M of all examined metals compared to control (Fig. 4). Moreover, the severe reduction in relative transcript levels of tax-2, tax-4, ceh-14 could be further observed in nematodes exposed to 50 and 100 M of all examined metals compared to control (D. Wang, personal communication). Thermosensory signal transduction requires the function of a cyclic nucleotide-gated channel encoded by tax-2 and tax-4 genes (Cobum and Bargmann, 1996). The ceh-14 LIM
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homeobox gene is required for the correct function of the AFD neurons (Cassata et al., 2000). Thus, metal exposure will influence the expression of most genes required for the differentiation and function of AFD neurons. That is, metal exposure at high concentrations will cause severe damage on the molecular basis for differentiation and function of AFD neurons. In addition, according to the observations in this study, the endpoint of relative fluorescent intensity of sensory ending in AFD neuron may be more suitable and sensitive for evaluating the neurotoxicity on thermotaxis from metal exposure. Exposure to all examined four metals at the concentration of 50 M caused a significant reduction of relative fluorescent intensities of sensory endings in AFDL neurons (Fig. 3). In contrast, the significant reduction of relative sizes of cell body fluorescent puncta in AFD neurons could be observed in nematodes exposed to 50 M of Hg, Ag, and Cr (Fig. 2). Especially, the severe decrease in relative intensities of cell bodies and relative lengths of sensory endings of AFD neurons could only be detected in nematodes exposed to 50 M of Hg and Cr (Figs. 2 and 3). 5. Conclusion Overall, our data suggest that metal exposure at higher concentrations can cause severe deficits in thermotaxis to cultivation temperature. Moreover, the morphology or development of AFD sensory neurons will be severely disrupted by high level of metal exposure, and the mainly altered structures by metal exposure are cell body and sensory ending in AFD neurons. In addition, metal exposure at high concentrations will result in the severe damage on the molecular basis for differentiation and function of AFD neuron in nematodes. Conflict of interest None. Acknowledgements Strains used in this study were provided by the Caenorhabdits Genetics Center (funded by the NIH National Center for Research Resource, USA). This work was supported by the grants from the National Natural Science Foundation of China (No. 30771113, 30870810) and the Program for New Century Excellent Talents in University. References Anderson, G.L., Boyd, W.A., Williams, P.L., 2001. Assessment of sublethal endpoints for toxicity testing with the nematode Caenorhabditis elegans. Environ. Toxicol. Chem. 20, 833–838. Anderson, G.L., Cole, R.D., Williams, P.L., 2004. Assessing behavioral toxicity with Caenorhabditis elegans. Environ. Toxicol. Chem. 23, 1235–1240. Boyd, W.A., Cole, R.D., Anderson, G.L., Williams, P.L., 2003. The effects of metals and food availability on the behavior of Caenorhabditis elegans. Environ. Toxicol. Chem. 22, 3049–3055. Brenner, S., 1974. The genetics of Caenorhabditis elegans. Genetics 77, 71–94. Cassata, G., Kagoshima, H., Andachi, Y., Kohara, Y., Durrenberger, M.B., Hall, D.H., Burglin, T.R., 2000. The LIM homeobox gene ceh-14 confers thermosensory function to the AFD neurons in Caenorhabditis elegans. Neuron 25, 587–597. Cobum, C., Bargmann, C.I., 1996. A putative cyclic nucleotide-gated channel is required for sensory development and function in C. elegans. Neuron 17, 695–706. Cole, R.D., Anderson, G.L., Williams, P.L., 2004. The nematode Caenorhabditis elegans as a model of organophosphate-induced mammalian neurotoxicity. Toxicol. Appl. Pharmacol. 194, 248–256. Dhawan, R., Dusenbery, D.B., Williams, P.L., 2000. A comparison of metal-induced lethality and behavioral responses in the nematode Caenorhabditis elegans. Environ. Toxicol. Chem. 19, 3061–3067. Donkin, S., Williams, P.L., 1995. Influence of developmental stage, salts and food presence on various end points using Caenorhabditis elegans for aquatic toxicity testing. Environ. Toxicol. Chem. 14, 2139–2147.
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Exposure to metals induces morphological and functional alteration of AFD neurons in nematode Caenorhabditis elegans Xiaojuan Xing, Min Du, Xuemei Xu, Qi Rui, Dayong Wang ∗ Key Laboratory of Developmental Genes and Human Disease in Ministry of Education, Department of Genetics and Developmental Biology, Southeast University Medical School, Nanjing 210009, China
a r t i c l e
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Article history: Received 5 December 2008 Received in revised form 4 March 2009 Accepted 4 March 2009 Available online 17 March 2009 Keywords: Metal exposure Thermotaxis AFD neuron ttx-1 Caenorhabditis elegans
a b s t r a c t Previous studies have revealed that metal exposure will cause severe deficits in perception behaviors. Here we investigated the effects of metal (Hg, Cu, Ag, and Cr) exposure on thermotaxis to cultivation temperature in Caenorhabditis elegans. Our data suggest that exposure to higher concentrations of examined metals induced severe deficits in thermotaxis, and a significant reduction in thermotaxis could be even observed in nematodes exposed to 2.5 M of Hg. Moreover, exposure to higher concentrations of examined metals and 2.5 M of Hg induced significant decreases in relative intensities and relative sizes of fluorescent puncta of cell bodies in AFD thermosensory neurons. In addition, exposure to higher concentrations of examined metals resulted in a significant reduction in relative intensities and relative lengths of sensory endings in AFD neurons. Furthermore, the relative transcript levels of ttx-1, which functions in specifying the fate of AFD neuron, were significantly decreased in nematodes exposed to 2.5 M of Hg, and 50 and 100 M of examined metals. Thus, metal exposure at high concentrations will induce the severe deficits in thermotaxis to cultivation temperature possibly by altering the morphology or development of AFD neuron and damaging the molecular basis for function of AFD neuron in nematodes. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Nematode Caenorhabditis elegans, a model animal best characterized at the genetic, physiological, molecular, and developmental levels (Riddle et al., 1997), has already been explored as a valuable bioindicator (Williams and Dusenbery, 1990a; Donkin and Williams, 1995). In addition, it has been proved that C. elegans can serve as an excellent candidate for studying the neurotoxicology, since it has only 302 neurons and the complete writing diagram for chemical and electrical connections is available (White et al., 1986; Leung et al., 2008). C. elegans can be used to test the environmental contaminants known to have neural toxic effects. The deficits in movement behavior can be monitored using a computer tracking system after exposure to metals or organophosphorus insecticides in nematodes (Dhawan et al., 2000; Anderson et al., 2001; Boyd et al., 2003; Anderson et al., 2004; Rajini et al., 2008). The toxic effects of metal exposure on endpoints of head thrash, body bend, and basic movements (forward sinusoidal movement (forward turn), reversal movement (backward turn), and turn) have been examined in nematodes (Wang et al., 2007a,b; Wang and Xing, 2008; Wang and Wang, 2008a,b). C. elegans was also used for the test of organophosphate-induced neurotoxicity by anticholinesterase
∗ Corresponding author. Tel.: +86 25 83272314 817; fax: +86 25 83324887. E-mail address: [email protected] (D. Wang). 1382-6689/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2009.03.006
activity assay (Cole et al., 2004; Rajini et al., 2008). Furthermore, exposure to metals of Al or Pb at high concentrations could induce severe decrease in memory behaviors (Ye et al., 2008a,b). Previous studies have revealed that metal exposure at higher concentrations will result in severe deficits in color vision, hearing ability, smelling ability, and olfactory function (Rose et al., 1992; Sulkowski et al., 2000; Wu et al., 2000; Urban et al., 2003; Gobba and Cavalleri, 2003). Thermotaxis is based on the thermal sense, which is an important perception behavior for the life of the animals (Mori and Ohshima, 1997). Temperature is a critical modulator of metabolism and behavior, and sensed by C. elegans using the AFD thermosensory neurons (Satterlee et al., 2001). In addition, nematodes grown in the presence of food at a particular temperature will actively seek this temperature when presented with a thermal gradient but will avoid this temperature if it is associated with an absence of food (Hedgecock and Russell, 1975; Mori, 1999). ttx-1, a homolog of otd/Otx, specifies the thermotaxis neuron fate in C. elegans, and misexpression of ttx-1 can convert other sensory neurons to an AFD-like fate (Satterlee et al., 2001). Thus, one of the objectives of this study was to investigate the possible effects of metal exposure on the thermotaxis to cultivation temperature in C. elegans. Four metals were selected, and they were Cr, Cu, Hg, and Ag, respectively. In addition, our specific aims were to examine the effects of metal exposure on the structures or morphologies of AFD sensory neurons and the expression patterns of ttx-1 gene in nematodes. The morphological changes of cell body
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and sensory ending of AFD thermosensory neurons and expression patterns of ttx-1 gene were examined in control and metal exposed nematodes.
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of ttx-1 was expressed as the ratio of ttx-1/act-1. At lease three replicates were performed for statistical purposes. 2.5. Statistical analysis
2. Materials and methods 2.1. Reagents and strains The metal concentrations used in this study were selected as previously described (Wang and Yang, 2007). Four concentrations of CrCl2 , CuSO4 , HgCl2, and AgNO3 solutions were used in the current work, and they were 2.5, 50, 100 and 150 M, respectively. Metal concentrations of exposed solutions were analyzed by atomic absorption spectrophotometry (AAS; Pye-Unicam model SP9, Cambridge, UK). All the chemicals were obtained from Sigma–Aldrich (St. Louis, MO, USA). Nematodes used in the present study were wild-type N2 and DA1267 (lin15(n765); dEx1267[lin-15(+) gcy-8::GFP]) labeling the AFD thermosensory neuron, originally obtained from the Caenorhabditis Genetics Center (funded by the NIH National Center for Research Resource, USA). They were maintained on nematode growth medium (NGM) plates seeded with Escherichia coli OP50 at 20 ◦ C as described (Brenner, 1974). Gravid nematodes were washed off the plates into centrifuge tubes, and were lysed with a bleaching mixture (0.45N NaOH, 2% HOCl). Age synchronous populations of larva (L4-stage) were obtained by the collection as described (Donkin and Williams, 1995). The L4-larva stage nematodes were washed with doubledistilled 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, 1990b). Exposures were performed in 12-well sterile tissue culture plates as described (Mutwakil et al., 1997). All exposures were 24-h long and were carried out in 20 ◦ C incubator in the presence of food. 2.2. Thermotaxis assay The method was performed as described (Gomez et al., 2001; Ye et al., 2008a). Approximately 50 nematodes were grown at 20 ◦ C overnight in the presence of a fresh lawn of the bacteria strain of OP50 on a 6 cm Petri dish. Young adults were transferred on to a fresh plate devoid of bacteria for 2 min, and individual nematodes were then deposited on a 9 cm Petri dish. A radial gradient of temperature was created by placing a vial containing frozen acetic acid on the bottom of the plate and incubating at 25 ◦ C for 90 min in the presence of a constant humidity of 60%. Upon removal of the nematodes from the plate, tracks left were photographed and analyzed. A trace is considered as isothermal if more than half of the trace length left on the agar surface by a single nematode is circular or presents an arc of circle near the isotherm of the cultivation temperature. Each data point represents 10 independent assays. 2.3. Fluorescence quantification To quantify fluorescence intensities, fluorescence images of neurons in control and metal exposed young adult nematodes were captured with a Zeiss Axiocam MRm camera on a Zeiss Axioplan 2 Imaging System with ×40 objective using SlideBook software (Intelligent Imaging Innovations). Images were acquired with a Quantix cooled CCD camera, and illumination was provided by a 175 W xenon arc lamp and Green Fluorescent Protein (GFP) filter sets. Exposure times were chosen to fill the 12-bit dynamic range without saturation, and out-of-focus light was removed with a constrained iterative deconvolution algorithm. Background fluorescence from the coverslip and from nonspecific tissue autofluorescence was removed by subtracting an image filtered with a low pass Gaussian filter. The puncta sizes for cell bodies or relative lengths of sensory endings were measured as the maximum radius for assayed fluorescent puncta. The relative fluorescence intensity of particular fusion proteins at the cell bodies or sensory endings was obtained by integrating pixel intensity in at least 20 nematodes. Similarly, the relative sizes of fluorescent puncta for cell bodies or relative lengths of sensory endings were also examined in at least 20 nematodes. The images were photographed and examined on the same day to avoid effects of light source variance on fluorescence intensity. 2.4. Semi-quantitative Reverse Transcriptase (RT)–Polymerase Chain Reaction (PCR) assay Total RNA was isolated using guanidinium thiocyanate/phenol method as described (Morse and Bass, 1999). Control and metal exposed nematodes were sampled for the RT–PCR assay. The frozen nematode pellets were harvested from 1 L of mixed stage liquid cultures. Purified Poly (A)+ RNA through two rounds of selection on oligo(dT)-cellulose was used for the RNA blotting. Genespecific primers were designed for ttx-1 (ttx-1F, 5 -ATGTCCTTGACGTCTTCC-3 , and ttx-1R, 5 -TTCCGTCCAAAGTGTAGG-3 ). Act-1 was used to determine the equal loading for each sample, and primers specific for act-1 were used in control reactions (act-1F, 5 -CGAAGCTTACCGTCCCAATCTACGAAG-3 ; act-1R, 5 TGAGAATTCGAAGCACTTGCGGTGAAC-3 ). Amplification of all DNA fragments for RT–PCR was performed for 30 cycles in a Perkin-Elmer 480 thermal cycler using a 55 ◦ C annealing temperature and a 1-min extension. The relative transcript level
All data in this article were expressed as means ± SD. Graphs were generated using Microsoft Excel (Microsoft Corp., Redmond, WA). One-way 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. Effects of metal exposure at different concentrations on the thermotaxis to cultivation temperature in C. elegans We first investigated the thermotaxis behaviors in nematodes exposed to Hg, Cu, Ag, and Cr at concentrations from 2.5 to 150 M. As shown in Fig. 1, exposure to 100 M (Cu, P < 0.05; Hg, Ag, and Cr, P < 0.01) and 150 M (P < 0.01) of all examined metals caused the significant decrease in percentage of nematodes performing isothermal tracking (IT) behavior after overnight feeding at 20 ◦ C. Exposure to 50 M of Hg (P < 0.01) and Cr (P < 0.05) could also result in the severe defects of thermotaxis to cultivation temperature, whereas exposure to 50 M of Ag and Cu did not alter the thermotaxis to cultivation temperature of exposed nematodes. Moreover, a significant (P < 0.05) reduction in thermotaxis to cultivation temperature could even be observed in nematodes exposed to 2.5 M of Hg. Therefore, exposure to higher concentrations of metals will induce severe deficits in thermotaxis to cultivation temperature, and the exposure to Hg can induce the most severe neurotoxicity on thermotaxis among the examined metals in C. elegans. 3.2. Effects of metal exposure at different concentrations on the cell body morphology of AFD sensory neuron in C. elegans Thermotaxis to cultivation temperature was under the control of AFD sensory neuron (Satterlee et al., 2001). Pgcy-8::GFP is a specific fluorescent marker to label the AFD sensory neuron (Satterlee et al., 2001). As shown in Fig. 2A and B, metal exposure could significantly change the relative fluorescent intensities of cell bodies in AFD neurons. Exposure to 100 M (Cu, P < 0.05; Hg, Ag, and Cr, P < 0.01) and 150 M (P < 0.01) of all examined metals caused the significant decrease in relative intensities of cell bodies in AFD neurons compared to control. Exposure to 50 M of Hg (P < 0.01) and Cr (P < 0.05) also induced a severe reduction in relative intensities of cell bodies in AFD neurons, whereas no remarkable alterations of relative intensities of cell bodies in AFD neurons could be observed in nematodes exposed to 50 M of Cu and Ag. In addition, a significant (P < 0.01) decrease in relative intensities of cell bodies in AFD neurons was further found in nematodes exposed to 2.5 M of Hg. No significant differences of relative intensities for cell bodies could be detected between AFDL and AFDR neurons in control and metal exposed nematodes. We also examined the effects of metal exposure at different concentrations on relative sizes of fluorescent puncta of cell bodies in AFD neurons. As shown in Fig. 2A and C, exposure to 100 and 150 M of all examined metals caused significant (P < 0.01) decrease in relative sizes of fluorescent puncta of cell bodies in AFD neurons compared to control. Moreover, exposure to 50 M of Hg, Ag, and Cr further induced the significant (P < 0.01) reduction of relative sizes of cell body fluorescent puncta in AFD neurons. In addition, exposure to 2.5 M of Hg also resulted in a significant (P < 0.01) decrease in relative sizes of cell body fluorescent puncta in AFD neurons. Similarly, no significant differences of relative sizes of cell body fluorescent puncta could be detected between AFDL and AFDR neurons in control and metal exposed nematodes. There-
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Fig. 1. . Effects of metal exposure at different concentrations on thermotaxis in C. elegans. Metals of Hg, Cu, Ag, and Cr were selected for the assay. Thermotaxis was evaluated by the percentage of nematodes performing isothermal tracking (IT) behavior after overnight feeding at 20 ◦ C. A trace is considered as isothermal if more than half of the trace length left on the agar surface by a single nematode is circular or present an arc of circle near the isotherm of the growth temperature. Bars represent means ± S.D. *P < 0.05 vs. 0 M; **P < 0.01 vs. 0 M.
fore, the morphology of cell bodies in AFD neurons can be severely altered by metal exposure to different degrees. 3.3. Effects of metal exposure at different concentrations on the sensory ending of AFD neuron in C. elegans Sensory cilia provide a compartment for the localization and exposure of signal transduction proteins to incoming sensory signals (Swoboda et al., 2000). AFD is a thermosensory neuron with a cilium exposed to the environment, and the finger-like microvilli can be observed at the end of the AFD dendrites (Swoboda et al., 2000). We next investigated the effects of metal exposure on the relative fluorescent intensities of sensory endings in AFDL neurons (Fig. 3A and B). The sensory endings of the AFD neurons are typically elaborate, consisting of a single cilium and numerous microvillar “fingers” (Fig. 3A). We found that, very different from the effects on morphology of cell body, exposure to metals of Hg, Cu, Ag, and Cr at concentrations from 50 to 150 M all caused a significant (P < 0.01) reduction of relative fluorescent intensities of sensory endings in AFDL neurons compared to control. Moreover, the severe (P < 0.01) decrease in relative fluorescent intensity of sensory ending in AFDL neuron was also detected in nematodes exposed to 2.5 M of Hg. In addition, no significant differences of relative fluorescent intensities of sensory endings could be detected between AFDL and AFDR neurons in control and metal exposed nematodes (data not shown). We further examined the effects of metal exposure on the relative lengths of sensory endings of AFDL neurons. As shown in Fig. 3A and C, exposure to 100 and 150 M of all examined metals resulted in the severe (P < 0.01) decrease in relative lengths of sensory endings in AFDL neurons compared to control. Furthermore, a significant reduction in relative lengths of sensory endings of AFDL neurons could be observed in nematodes exposed to 50 M of Hg (P < 0.01) and Cr (P < 0.05). In contrast, no significant alteration of relative lengths of sensory endings in AFDL neurons could be detected in nematodes exposed to 50 M of Cu and Ag, as well as to 2.5 M of all examined metals, compared to control. Similarly, no significant differences of relative lengths of sensory endings could be detected between AFDL and AFDR neurons in control and metal exposed nematodes (data not shown). In addition, no obvious
defects of dendrites and axon guidance were observed in nematodes exposed to all examined metals (data not shown). Therefore, metal exposure will alter the morphology and development or suppress the extension or growth of sensory ending of AFD cilium. 3.4. Effects of metal exposure at different concentrations on the expression levels of ttx-1 gene in C. elegans ttx-1 regulates all differentiated characteristics of the AFD neurons, and mutations of ttx-1 affect AFD neuron functions (Satterlee et al., 2001). We finally examined the changes of transcript levels of ttx-1 in control and metal exposed nematodes as revealed by RT–PCR assay. Because exposure to a very high concentration (150 M) of examined metals would severely affect the locomotion behaviors of exposed nematodes (data not shown), we only investigated the expression patterns of ttx-1 in control and metal exposed nematodes at concentrations not more than 100 M. As shown in Fig. 4, exposure to 2.5 M of Hg and Cr could induce the significant (P < 0.01) decrease in relative transcript levels of ttx-1 compared to control, but exposure to Cu and Ag at the same concentration would not affect the transcript levels of ttx-1 in exposed animals. Moreover, the significant decrease in relative transcript levels of ttx-1 could also be observed in nematodes exposed to all examined metals at concentrations of both 50 M (Cu, P < 0.05; Hg, Ag, and Cr, P < 0.01) and 100 M (P < 0.01) compared to control. The Northern blotting results were consistent with the observations by RT–PCR assay (data not shown). Therefore, metal exposure may result in the damage on the molecular basis for AFD neuron function to different degrees. 4. Discussion Perception to environmental cues is an important behavior for animals and human beings. Previous studies have revealed the possible effects of metal exposure on perception behaviors in animals. For example, exposure to mercury vapor can induce sub-clinical color vision impairment (Urban et al., 2003; Gobba and Cavalleri, 2003). A significant correlation between a high, long-term lead exposure index (defined by duration of employment and ambient
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Fig. 2. . Effects of metal exposure at different concentrations on morphology of AFD sensory neuron in C. elegans. Metals of Hg, Cu, Ag, and Cr were selected for the assay. (A) Morphological patterns of AFD sensory neurons. (B) Quantification of relative fluorescent intensities in cell bodies of AFD sensory neurons. (C) Relative sizes of fluorescent puncta for cell bodies of AFD sensory neuron. Pgcy-8::GFP labels the AFD sensory neurons. “L” indicates the cell body of left neuron; “R” indicates the cell body of right neuron. Bars represent means ± S.D. *P < 0.05 vs. 0 M; **P < 0.01 vs. 0 M.
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Fig. 3. . Effects of metal exposure on sensory ending (cilium) of AFD sensory neuron in C. elegans. Metals of Hg, Cu, Ag, and Cr were selected for the assay. (A) Morphological patterns of AFDL (the left one of paired AFD neurons) sensory neurons in nematodes exposed to metals. (B) Quantification of relative lengths of sensory endings of AFDL sensory neurons. (C) Relative sizes of fluorescent puncta for sensory endings of AFDL sensory neurons. Pgcy-8::GFP labels the AFD sensory neuron. Con, control without metal exposure. Bars represent means ± S.D. *P < 0.05 vs. 0 M; **P < 0.01 vs. 0 M.
lead concentration) and decreased hearing ability was found in two lead-battery manufacturing factories, suggesting that lead exposure might precipitate a more severe auditory than noise-exposure effect (Wu et al., 2000). Smell impairment caused by occupationally exposure to cadmium was also observed in workers (Sulkowski et al., 2000). Chronic occupational cadmium exposure sufficient to
cause renal damage is also associated with impairment in olfactory function (Rose et al., 1992). Although no changes in olfactory function as measured by either absolute threshold or two-odor discrimination tasks, nickel exposure could result in selective lesions to the olfactory system and decreases in the number of bipolar sensory receptor cells, the thickness of olfactory epithelium, and
Fig. 4. . Expression pattern changes of ttx-1 induced by metal exposure in C. elegans. Metals of Hg, Cu, Ag, and Cr were selected for the assay. (A) Semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction (RT–PCR) assay of expression patterns of ttx-1 gene. Act-1 was used to determine the equal loading for each sample. (B) Changes of relative transcript levels of ttx-1 in control and metal exposed nematodes. Bars represent means ± S.D. *P < 0.05 vs. 0 M; **P < 0.01 vs. 0 M.
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carnosine levels in rats (Evans et al., 1995). Multiple linear regression analysis relating to lead workers’ test performance and their lead exposure showed that performance on the sensory store memory test was significantly related to lead exposure (Williamson and Teo, 1986). Nevertheless, seldom studies have been performed to analyze the effects of metal exposure on the thermotaxis behaviors. In the present study, our data suggest that exposure to higher concentrations of our examined metals would induce severe deficits in thermotaxis to cultivation temperature, and the exposure to Hg could induce the most severe neurotoxicity on thermotaxis among the examined metals (Fig. 1). Exposure to higher concentrations of other heavy metals, such as Pb and Mn, also induced the severe neurotoxicity on thermotaxis in nematodes (D. Wang, personal communication). These observations further support the notion that exposure to high levels of toxic heavy metals may induce severe deficits in perception behaviors in animals. AFD sensory neuron plays a pivotal role in regulating the thermotaxis to cultivation temperature in nematodes (Mori, 1999; Satterlee et al., 2001). Our data suggest that morphologies or development of both the cell body and the sensory ending of AFD neurons can be severely influenced by metal exposure at relatively higher concentrations. Exposure to 100 and 150 M of all examined metals caused the significant decrease in relative intensities and relative sizes of fluorescent puncta of cell bodies in AFD neurons compared to control (Fig. 2). Similarly, exposure to 100 and 150 M of all examined metals caused the significant decrease in relative intensities and relative lengths of sensory endings in AFD neurons compared to control (Fig. 3). Moreover, the severe defects of relative intensities and relative sizes of fluorescent puncta of cell bodies, as well as relative intensities and relative lengths of sensory endings, in AFD neurons could be further detected in nematodes exposed to 50 M of Hg and Cr (Figs. 2 and 3). Thus, the morphology of AFD thermosensory neurons will be severely disrupted and the development of AFD thermosensory neurons will be noticeably inhibited by high level of metal exposure. Considering the fact that no obvious defects of dendrites and axon guidance could be observed in nematodes exposed to all examined metals (data not shown), the mainly altered structures by metal exposure may be cell body and sensory ending in AFD neurons. In addition, no significant differences of relative lengths of sensory endings could be detected between AFDL and AFDR neurons in control and metal exposed nematodes (data not shown). Nevertheless, no phenotype of loss of cilium as previously observed in daf-19 mutants could be found in nematodes exposed to all examined metals (Swoboda et al., 2000), suggesting that metal exposure will not influence the cilium formation process. Furthermore, the significant decrease in relative intensities or relative sizes of fluorescent puncta of cell bodies in AFD neurons could be further observed in nematodes exposed to 2.5 M of Hg. The severe decrease in relative fluorescent intensities of sensory endings in AFDL neurons could also be detected in nematodes exposed to 2.5 M of Hg. Together with the effects from exposure to 2.5 M of Hg on thermotaxis to cultivation temperature, our results demonstrated that Hg exposure can activate the most severe neurotoxicity on thermotaxis to cultivation temperature among the examined metals. ttx-1 functions is used in specifying the thermotaxis neuron fate, and is expressed in the AFD neurons (Satterlee et al., 2001). The significant decrease in relative transcript levels of ttx-1 could be detected in nematodes exposed to 2.5 M of Hg, and 50 and 100 M of all examined metals compared to control (Fig. 4). Moreover, the severe reduction in relative transcript levels of tax-2, tax-4, ceh-14 could be further observed in nematodes exposed to 50 and 100 M of all examined metals compared to control (D. Wang, personal communication). Thermosensory signal transduction requires the function of a cyclic nucleotide-gated channel encoded by tax-2 and tax-4 genes (Cobum and Bargmann, 1996). The ceh-14 LIM
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homeobox gene is required for the correct function of the AFD neurons (Cassata et al., 2000). Thus, metal exposure will influence the expression of most genes required for the differentiation and function of AFD neurons. That is, metal exposure at high concentrations will cause severe damage on the molecular basis for differentiation and function of AFD neurons. In addition, according to the observations in this study, the endpoint of relative fluorescent intensity of sensory ending in AFD neuron may be more suitable and sensitive for evaluating the neurotoxicity on thermotaxis from metal exposure. Exposure to all examined four metals at the concentration of 50 M caused a significant reduction of relative fluorescent intensities of sensory endings in AFDL neurons (Fig. 3). In contrast, the significant reduction of relative sizes of cell body fluorescent puncta in AFD neurons could be observed in nematodes exposed to 50 M of Hg, Ag, and Cr (Fig. 2). Especially, the severe decrease in relative intensities of cell bodies and relative lengths of sensory endings of AFD neurons could only be detected in nematodes exposed to 50 M of Hg and Cr (Figs. 2 and 3). 5. Conclusion Overall, our data suggest that metal exposure at higher concentrations can cause severe deficits in thermotaxis to cultivation temperature. Moreover, the morphology or development of AFD sensory neurons will be severely disrupted by high level of metal exposure, and the mainly altered structures by metal exposure are cell body and sensory ending in AFD neurons. In addition, metal exposure at high concentrations will result in the severe damage on the molecular basis for differentiation and function of AFD neuron in nematodes. Conflict of interest None. Acknowledgements Strains used in this study were provided by the Caenorhabdits Genetics Center (funded by the NIH National Center for Research Resource, USA). This work was supported by the grants from the National Natural Science Foundation of China (No. 30771113, 30870810) and the Program for New Century Excellent Talents in University. References Anderson, G.L., Boyd, W.A., Williams, P.L., 2001. Assessment of sublethal endpoints for toxicity testing with the nematode Caenorhabditis elegans. Environ. Toxicol. Chem. 20, 833–838. Anderson, G.L., Cole, R.D., Williams, P.L., 2004. Assessing behavioral toxicity with Caenorhabditis elegans. Environ. Toxicol. Chem. 23, 1235–1240. Boyd, W.A., Cole, R.D., Anderson, G.L., Williams, P.L., 2003. The effects of metals and food availability on the behavior of Caenorhabditis elegans. Environ. Toxicol. Chem. 22, 3049–3055. Brenner, S., 1974. The genetics of Caenorhabditis elegans. Genetics 77, 71–94. Cassata, G., Kagoshima, H., Andachi, Y., Kohara, Y., Durrenberger, M.B., Hall, D.H., Burglin, T.R., 2000. The LIM homeobox gene ceh-14 confers thermosensory function to the AFD neurons in Caenorhabditis elegans. Neuron 25, 587–597. Cobum, C., Bargmann, C.I., 1996. A putative cyclic nucleotide-gated channel is required for sensory development and function in C. elegans. Neuron 17, 695–706. Cole, R.D., Anderson, G.L., Williams, P.L., 2004. The nematode Caenorhabditis elegans as a model of organophosphate-induced mammalian neurotoxicity. Toxicol. Appl. Pharmacol. 194, 248–256. Dhawan, R., Dusenbery, D.B., Williams, P.L., 2000. A comparison of metal-induced lethality and behavioral responses in the nematode Caenorhabditis elegans. Environ. Toxicol. Chem. 19, 3061–3067. Donkin, S., Williams, P.L., 1995. Influence of developmental stage, salts and food presence on various end points using Caenorhabditis elegans for aquatic toxicity testing. Environ. Toxicol. Chem. 14, 2139–2147.
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