Transgenerational effects of heavy metals on L3 larva of Caenorhabditis elegans with greater behavior and growth inhibitions in the progeny

Ecotoxicology and Environmental Safety 88 (2013) 178–184

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Transgenerational effects of heavy metals on L3 larva of Caenorhabditis elegans with greater behavior and growth inhibitions in the progeny ZhenYang Yu, XiaoXue Chen, Jing Zhang, Rui Wang, DaQiang Yin n Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 October 2012 Received in revised form 9 November 2012 Accepted 10 November 2012 Available online 4 December 2012

Heavy metals are ubiquitous environmental pollutants, and their toxic effects have been widely studied. However, their transgenerational effects between parent and progeny at environmental relevant concentrations need further investigations. Currently, L3 stage of Caenorhabditis elegans was exposed to aqueous metals (Cd, Cu, Pb and Zn) at environmentally realistic concentrations for 96 h. The whole exposure time covered the formation of sperm, ovum and eggs. Subsequently the behavior and growth indicators were measured. The parent nematodes were then bleached to gain synchronized eggs, which were cultured under non-toxic conditions to L3 stage when the same indicators were measured in the progeny. The parent suffered concentration-dependent inhibitions on behavior and growth. Based on the median effective concentration (EC50) values, body bending frequency showed relatively higher sensitivity than other behavior indicators. The inhibitions on growth and behavior of progeny were more severe than those of the parent, based on their respective EC50 values. Interestingly, Cd was not the most toxic metal in either parent or progeny according to EC50 values, but its EC50 ratios between parent and progeny (EC50, parent/EC50, progeny) were the most significant, indicating its greatest transgenerational effects. The results demonstrated the higher sensitivity of L3 larva stage of C. elegans in the transgenerational effect studies than other life stages used before. Our findings suggested that parental exposure to heavy metals can multiply their harmful effects in following generations. & 2012 Elsevier Inc. All rights reserved.

Keywords: Heavy metal Transgenerational effect Caenorhabditis elegans L3 stage Behavior Growth

1. Introduction Heavy metals are ubiquitous pollutants worldwide. Due to their persistence and accumulation in the environment, and their bio-magnification in organisms via food chain transfer, they are gaining more ecological and public concerns (Cain et al., 2011; Yi et al., 2011). Accordingly, their adverse effects have been widely studied. They can cause death, growth and behavior inhibitions, and oxidative stresses on numerous organisms (Arambaˇsic´ et al., 1995; IARC, 1997; Dhawan et al., 2000; Anderson et al., 2001; Boyd et al., 2003; Pinho and Bianchini, 2010). However, most of the effects were observed within one generation of the test organism, while the transgenerational effects are increasingly recognized as one critical aspect of toxicity studies. The phrase of transgenerational effects means that the parental experiences in critical development stages have influences on the phenotype, behavior and even health/disease of the progeny. Thus, the transgenerational effects are referred as ‘‘the developmental origins of health and disease’’ (Swanson et al., 2009; Zeng et al., 2011), and therefore can be used to indicate the

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0147-6513/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2012.11.012

long-term consequences. Heavy metals had been reported to provoke transgenerational effects. Exposure to copper (Cu) during the parental development stages of Protophormia terraenovae (fly) resulted in immune responses which were even apparent in the ¨ offspring that was not exposed to the heavy metal (Polkki et al., 2012). Although the study considered the influence of early parental development, it only employed one exposure concentration, which was difficult to draw any concentration-dependent conclusions. The transgenerational effects of cobalt (Co) (Wang et al., 2007b), ion (Fe) (Hu et al., 2008), nickel (Ni) (Wang and Wang, 2008), lead (Pb) (Wang and Yang, 2007) and zinc (Zn) (Wang et al., 2007a) have been demonstrated by the decreased locomotion and body sizes in the unexposed offspring of parentally exposed Caenorhabditis elegans (nematode). Although these studies considered multiple concentrations in the experiments, they employed L4 larva stage (except an un-identified life stage in the study on Ni), which is not persuasive enough to represent the early parental development according to the life cycle of C. elegans. The nematode C. elegans is a good animal model for ecotoxicological studies because of its abundance in soil ecosystem, it is convenient handling in the laboratory, and its relative high sensitivity to different kinds of stresses or toxicants (Leung et al.,

Z. Yu et al. / Ecotoxicology and Environmental Safety 88 (2013) 178–184

2008). This free living nematode is mostly single self-fertilizing hermaphrodite with an occasional rare male (Blaxter and Denver, 2012). It first produces sperm at L4 stage and then stores it to fertilize the eggs that develop later in development (Hill et al., 2006). Therefore, the aforementioned exposure at L4 stage may not include all of the sperm production. Instead, L3 stage of C. elegans will serve better to represent the early parental development, and should provoke greater transgenerational effects on the progeny. Various indicators of C. elegans have been used to elucidate the toxicity of heavy metals. A common approach to assess the health status in C. elegans is to measure its locomotion behavior (de Bono and Maricq, 2005). As reviewed by Leung et al. (2008), a defect in the nematode locomotion reflects an impairment of the neuronal network. One recent study also demonstrated the correlation (R2 ¼0.969, Po0.01) between behavior (body bend) and reactive oxygen species (ROS) production (Wu et al., 2012b). Another general of nematode health parameter is the body length (body size or growth), which has been widely used in earlier reports (Anderson et al., 2001; Boyd et al., 2003; Wang et al., 2007b; Yu et al., 2012b). In the present study, four typical heavy metals, cadmium (Cd), Cu, Pb and Zn, were chosen as tested chemicals. These four heavy metals usually paralleled in their occurrences and even overloads in water and sediments, and their environmental concentrations were usually at mmol/L and mmol/L (Zheng et al., 2008; Suresh et al., 2012; Wang et al., 2012; Yu et al., 2012a). Currently, L3 stage of C. elegans was exposed to aqueous metals (Cu, Pb, Cd and Zn) at environmentally realistic concentrations for 96 h, and then the behavior and growth inhibitions on the exposed parent (P0) were measured. The unexposed progeny (F1) was separated and the same indicators were measured. As expected, L3 stage of nematodes indeed showed higher sensitivity than L4 stage. The transgenerational effects of heavy metals at environmentally realistic concentrations indicated that parental exposure can multiply the harmful effects of heavy metal pollution in following generations.

2. Materials and methods 2.1. Tested chemicals Stock solutions of CuCl2, Pb(NO3)2, Cd(NO3)2  4H2O, and ZnCl2 were prepared with sterilized K-medium (Williams and Dusenbery, 1990). In an earlier report (Anderson et al., 2001), the median lethal concentration (LC50) value of Cu2 þ was 990 mmol/L, and the median effective concentration (EC50) values on movement were 170 mmol/L, and 47 mmol/L for Cd2 þ and Pb2 þ , respectively. Meanwhile, 200 mmol/L of Zn2 þ caused approximately 50 percent inhibition on body bends of C. elegans (Wang et al., 2007a). Based on the published data, the concentrations of each metal in the present study were selected on the attempt to avoid complete suppression on nematodes and to represent environmentally realistic concentrations at the same time. The concentrations were 0.022–220 mmol/L for Cd, 0.047–470 mmol/L for Cu, 0.0048–48.0 mmol/L for Pb, and 0.038–380 mmol/L for Zn. Each metal has five concentrations, which were diluted from corresponding stocking solutions with sterilized K-medium. The actual metal concentrations were determined by graphite furnace atomic absorption spectrometry (Perkin-Elmer, AA-600) (Santos et al., 2008; Yu et al., 2012b). 2.2. Preparation of nematode C. elegans (wild-type N2) and E. coli OP50 (the nematode food), both stocked on a nematode growth medium (NGM) agar at 4 1C in the dark, were kind gifts from Institute of Development Biology and Molecular Medicine, Fudan University, Shanghai, China. The preparation of nematodes were performed according to earlier reports (Brenner, 1974). First, E. coli OP50 was inoculated from the stocked NGM agar into sterile lysogeny broth (LB) culture medium (10 g tryptone, 5 g yeast extract and 10 g NaCl in 1 L distilled water, autoclaved at 121 1C for 20 min and cooled to room temperature before used). The bacteria were incubated with a shaking speed of 180 rpm at 37 1C for 24–48 h, during which the NGM agar was prepared as follows: (1) 3 g NaC1, 2.5 peptone and 17 g agar in 975 mL distilled water; (2) after autoclaving at 121 1C for 20 min, 1 mL cholesterol in ethanol (5 mg/mL), 1 mL M CaCl2, 1 mL M MgSO4, and 25 mL M K3PO4 buffer (pH 6.0) are

179

added in order, when the agar was 40–50 1C; (3) the NGM agar was poured into sterile Petri dishes (60 mm) to cool down to room temperature. E. coli OP50 suspensions (approximately 150–200 mL) were spread on the NGM agars, which were kept at 37 1C for 24 h to form bacterial lawn and was cooled down to 20 1C. Then, with the help of sterile pipette tip, one eighth of the stocked NGM agar, with nematodes on it, was cut off and transferred onto the freshly prepared NGM agars (8 in total) with the bacterial lawn. The 8 NGM agars with nematodes were kept in a 20 1C incubator for 4 d. On the fourth day, C. elegans was washed off each of the 8 NGM agars with 1.5 mL sterile water, and transferred to a 15 mL sterile centrifuge tube (approximately 12 mL in total). After 20–30 min allowing the nematodes to settle, 6 mL supernatants were deserted, and the nematodes in the left 6 mL were inoculated onto 30 freshly prepared NGM agars with the bacterial lawn (approximately 200 mL on each). After incubation at 20 1C for another 4 d, nematodes on the 30 NGM agars were inoculated onto 120 NGM agars with the bacterial lawn in the same way. When nematodes on the 120 NGM agars were incubated for 4 d, they were inoculated onto 480 NGM agars with the bacterial lawn. Following incubation at 20 1C for 2 d, the nematodes were ready for age synchronization. The gravid nematodes and newly produced eggs were washed off the 480 agars (2 mL sterile water for each agar) and collected into 48 sterile centrifuge tubes (15 mL in each). After 30 min allowing the nematodes to settle, 13 mL supernatants were deserted, and in each centrifuge tube were added 12 mL fresh Clorox solutions containing 0.5 M NaOH and 1 percent NaOCl (diluted from antiformin, 4–6 percent, Sinopharm Group Co. Ltd., China) (Emmons et al., 1979). After 10–15 min, the centrifuge tubes were centrifuged at 2500 rpm for 3 min at 20 1C. With supernatants deserted, 10 mL sterile water were added in each centrifuge tubes to wash the Clorox solution off the eggs, followed by a centrifugation at 2500 rpm for 3 min at 20 1C. The wash procedure was repeated twice. Then, the synchronized eggs in each tube were resuspended with 200 mL sterile K-medium and inoculated onto a NGM agar with the bacterial lawn (48 agars in total) for 36 h to obtain L3 nematodes (Van Gilst et al., 2005). The reason why L3 nematodes were chosen for toxicity tests was to cover the L4-adult molt, when the nematode sperm, ovum and eggs start to form (Hill et al., 2006), so as to provide a window for prenatal exposure. Before used, L3 nematodes were washed off the NGM agars and fasted in sterile K-medium for 2 h to digest the food in the guts (Reinke et al., 2010). 2.3. Toxicity experiment design The toxicity experiments were performed according to previous study with some modifications (Wang et al., 2007b). The brief steps are as follows. The exposure to the nematodes was performed in the 96-well sterile culture plates with 8 rows and 12 columns. Two heavy metals, five concentrations for each, were arranged in 10 columns with 8 wells as parallel for each concentration. K-medium was arranged in the left two columns as the controls. Then, L3 nematodes were added in the wells. Each test well typically contained 100 mL metal solutions and 100 mL K-medium containing 100 nematodes. All exposures lasted 96 h and were carried out in the absence of food at 20 1C. After the exposure, nematodes from 3 wells for each of the five metal concentrations and 6 wells of the control were collected into respective 1.5 mL centrifuge tubes. After 30 min allowing the nematodes to settle, the nematodes in the bottom were transferred into new 1.5 mL centrifuge tubes, where they were washed with 1 mL sterile water. After 30 min of settlement, the nematodes in the bottom were used for the indicator measurements of the exposed generation marked as P0. On the other hand, nematodes from the 5 remaining wells for each of the five concentrations and 10 wells of the control were collected into respective 1.5 mL centrifuge tubes (1 tube for each metal concentration and 2 tubes for the control). After 30 min allowing the nematodes to settle, the nematodes in the bottom were transferred into new 1.5 mL centrifuge tubes (1 tube for each treatment), where they were washed with 1 mL sterile water. After 30 min of settlement, the nematodes in the bottom were transferred to toxicant-free NGM agars with the bacterial lawn. The nematodes were allowed to grow for 36 h to produce enough eggs for the aforementioned age-synchronization (Emmons et al., 1979), after which the age-synchronized eggs were allowed to grow on NGM agars with the bacterial lawn for another 36 h. Then, the nematodes were collected into respective 1.5 mL centrifuge tubes. After 30 min allowing the nematodes to settle, the nematodes in the bottom were transferred into new 1.5 mL centrifuge tubes, where they were washed with 1 mL sterile water. After 30 min of settlement, the nematodes in the bottom were used for subsequent indicator measurements of the progeny (F1). The whole experiment was carried out independently in triplicate. 2.4. Growth and behavior indicator Growth and behavior indicators were determined and calculated according to our previous study (Yu et al., 2011, 2012b). Briefly, the nematodes were transferred on NGM agar without the bacterial lawn. After 2 h allowing water evaporate, the nematodes were captured with the dissecting microscope. The images captured by the dissecting microscope were used. A sequence of polylines was drawn following

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the nematodes’ midline from the head to the tail and back to the head. The body length was calculated as BL¼(length of the polylines)/2. At least 20 nematodes were repeated for each treatment in each experimental replicate. After the image capture for growth indicator, nematodes were immediately captured for the videos. Then nematodes were scored for the number of body bending frequency (BBF), reversal movement (RM) and Omega turn (OT) in an interval of 60 s. BBF was the times when the posterior bulb of the pharynx changed the direction along the vertical direction of the traveling path within 60 s. RM was counted as every time when the traveling direction changed including backward turns and OT. OT was referred as the movement when the head of the nematode touched its tail looking like the shape of the Greek letter Omega. At least 6 nematodes were examined for each treatment in each experimental replicate. 2.5. Statistical analysis Effects on growth (BL) and behavior indicators (BBF, RM and OT) in each treatment were all calculated as a percentage of that of the concurrent control (POC) (Anderson et al., 2001; Yu et al., 2012b). The effects on P0 or F1 used their own concurrent controls in the data calculation. One-way ANOVA (using Tukey test) was performed by Origin Pro 7.5 (Origin Lab Corp., USA), and the probability levels of 0.05 were considered statistically significant (p o 0.05). The ANOVA was carried out among concentration groups (including the control), and between parents and progeny. Linear regressions were calculated with the Origin Pro 7.5 to obtain EC50 values.

3. Results and discussion 3.1. Effects of heavy metals on the nematode parent The statistics of the fitted functions and median effective concentration (EC50) values for the effects of each heavy metal on the nematode parent (P0) are listed in Table 1. The values of R2 ( 40.7470) indicated the good relationships between the exposure concentrations of heavy metals and the inhibitions on the

body bending frequency (BBF) and body length (BL). The linear regression functions in Table 1 indicated the concentrationdependent effects. All the heavy metal exposed treatments caused significant inhibition effects (p o0.05) as compared to the control (BBF in Fig. 1, BL in Fig. 4), with the lowest percentages of controls (POCs) in the highest concentration. The EC50 values on BBF (Table 1) were 1.1E þ02, 2.3Eþ01, 4.1 and 7.7Eþ01 mmol/L for Cd, Cu, Pb and Zn, respectively. Therefore, the toxicities of heavy metals on BBF followed the order of Pb 4Cu4Zn 4Cd. The EC50 values on BL (Table 1) were 9.6Eþ01, 1.1Eþ01, 4.0 and 43.8Eþ02 mmol/L for Cd, Cu, Pb and Zn, respectively. Thus, the toxicities on BL followed the order of Pb 4Cu4Cd4Zn. Moreover, the EC50 values of BBF were lower than those of BL in Cd and Zn, while they were similar to those in Cu and Pb. The effects of heavy metals on reversal movement (RM) and Omega turn (OT) in P0 are shown in Figs. 2 and 3. In general, all the tested heavy metals evoked significant inhibition effects (po0.05) on RM and OT as compared to the control, in a concentration-dependent manner. However, the chosen concentrations of heavy metals in the current study caused no more than 50 percent of inhibition effects on RM and OT (POC values all higher than 50 percent), therefore the exact EC50 values could not be calculated (Table 1). In three behavioral indicators, BBF was the most sensitive, and this observation was different from previous report, where RM was the most sensitive when studying the effects of sulfamethoxazole on behavior of C. elegans (Yu et al., 2011). Since the nematode behavior is well connected with the neuronal network (Loria et al., 2004; Leung et al., 2008), such different results might indicate different toxicity mechanism of heavy metals and antibiotics on the nematode muscle and nervous system. In the present study, behavioral indicators did not show obvious superior sensitivities than growth indicator, as suggested

Table 1 The correlation coefficient (R2) of the fitted function, median effective concentrations (EC50), and the ratio of EC50, P0/EC50, metals on nematodes parent (P0, directly exposed) and progeny (F1, indirectly exposed) after 96 h prenatal exposure.

F1

for behavior and growth effects of heavy

Indicators

Chemicals

Functiona

R2

EC50 (mmol/L)

EC50,

Body bending frequency (BBF)

Cd

P0 F1 P0 F1 P0 F1 P0 F1 P0 F1 P0 F1 P0 F1 P0 F1 P0 F1 P0 F1 P0 F1 P0 F1 P0 F1 P0 F1 P0 F1 P0 F1

y¼  10.39xþ 71.32 y¼  12.92x þ63.15 y¼  9.91xþ 63.57 y¼  12.44x þ56.23 y¼  9.66xþ 59.83 y¼  12.71x þ49.56 y¼  12.60xþ 73.79 y¼  13.46x þ70.95 y¼  5.67xþ 87.94 y¼  8.21xþ 79.76 y¼  6.53xþ 81.46 y¼  9.07xþ 74.12 y¼  5.86x þ80.58 y¼  8.91xþ 70.31 y¼  7.43xþ 85.69 y¼  8.65xþ 80.81 y¼  5.89xþ 83.31 y¼  5.05xþ 82.22 y¼  5.04xþ 74.95 y¼  6.26xþ 70.17 y¼  4.02xþ 79.17 y¼  4.36xþ 75.55 y¼  5.94xþ 79.32 y¼  8.47xþ 71.74 y¼  11.48x þ72.76 y¼  11.72x þ55.03 y¼  15.38x þ66.20 y¼  15.40xþ 58.18 y¼  13.31x þ57.96 y¼  11.00xþ 47.29 y¼  9.23xþ 81.88 y¼  10.24xþ 68.59

0.9528 0.9248 0.7970 0.8042 0.8868 0.9093 0.9785 0.9713 0.9153 0.9739 0.8756 0.8540 0.8354 0.9122 0.9354 0.9169 0.9589 0.9094 0.5013 0.5422 0.6810 0.4988 0.7390 0.8565 0.9739 0.8432 0.9865 0.9608 0.9689 0.7475 0.9629 0.8957

1.1Eþ 02 1.0E þ 01 2.3Eþ 01 3.2Eþ 00 4.1Eþ 00 9.2E  01 7.7Eþ 01 3.6Eþ 01 42.2Eþ 02 42.2Eþ 02 44.7Eþ 02 44.7Eþ 02 44.8Eþ 01 44.8Eþ 01 43.8Eþ 02 43.8Eþ 02 42.2Eþ 02 42.2Eþ 02 44.7Eþ 02 44.7Eþ 02 44.8Eþ 01 44.8Eþ 01 43.8Eþ 02 43.8Eþ 02 9.6Eþ 01 2.7Eþ 00 1.1Eþ 01 3.4Eþ 00 4.0E þ 00 5.7E  01 43.8Eþ 02 6.5Eþ 01

Cu Pb Zn Reversal movement (RM)

Cd Cu Pb Zn

Omega turn (OT)

Cd Cu Pb Zn

Body length (BL)

Cd Cu Pb Zn

a

y: effects, calculated as the percentage of control (POC, %); x: log concentration, concentration unit was mmol/L before logarithm.

P0/EC50, F1

11.0 7.2 4.5 2.1 – – – – – – – – 35.6 3.2 7.0 45.8

Z. Yu et al. / Ecotoxicology and Environmental Safety 88 (2013) 178–184

Cu

Cd

Percentage of control

100

80

181

Zn

Pb **

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4.

l E03

Co nt ro

4. 8

Co nt ro 4. l 7E -0 2 4. 7E -0 4. 1 7E + 4. 00 7E +0 1 4. 7E +0 2

Co nt r 2. ol 2E -0 2. 2 2E -0 2. 1 2E + 2. 00 2E +0 1 2. 2E +0 2

0

Fig. 1. The effects of cadmium (Cd), copper (Cu), lead (Pb) and zinc (Zn) on the body bending frequency of the nematode parent (P0, blank) after prenatal exposure and the indirectly exposed nematode progeny (F1, shade). *: significantly different from the control, p o0.05; ]: significantly different from the lower concentration, p o0.05; þ : significantly different effects in progeny than in parent, p o 0.05.

in earlier studies on the effects of heavy metals (Wu et al., 2012a). Meanwhile, the EC50 values for behavior and growth indicators were generally lower than those in earlier reports (Dhawan et al., 2000; Anderson et al., 2001; Wang et al., 2007a; Wang and Yang, 2007; Wu et al., 2012a), which also covered a wide range of environmental concentrations. Such difference might be related to the different metal speciation of the tested metals, as well as the exposure time. Another explanation might be the varied choice of nematode life stages, with L1 larva in Wu et al. (2012a), L4 larva in Wang et al. (2007a) and Wang and Yang (2007), and adult in Anderson et al. (2001) and Dhawan et al. (2000), since nematodes in different life stages responded differently to the exposed stress (Darr and Fridovich, 1995; Guo et al., 2009). Thus, the lower EC50 results in the present study suggested the superior sensitivity of L3 larva stage in C. elegans. 3.2. Effects of heavy metals on the nematode progeny The statistics of the fitted functions and EC50 values for the effects of heavy metals on BBF and BL in the nematode progeny (F1) are also listed in Table 1. Good relationships (R2 40.7475) were found between the parental exposure concentrations and the effects on the BBF and BL in F1. The linear regression functions in Table 1 indicated the concentration-dependent transgenerational effects. The tested heavy metals all resulted in significant inhibition effects (p o0.05) as compared to the concurrent control even at the lowest concentration level (BBF in Fig. 1, BL in Fig. 4). In Table 1, the EC50 values on BBF were 1.0E þ01, 3.2, 9.2E-01 and 3.6Eþ 01 mmol/L for Cd, Cu, Pb and Zn, respectively. Thus, the toxicities of heavy metals on BBF in F1 followed the order of Pb4Cu 4Cd4Zn. Meanwhile, the EC50 values on BL (Table 1) were 2.7, 3.4, 5.7E-01 and 6.5E þ01 mmol/L, respectively. Therefore, the order of the toxicities was Pb4Cd4Cu 4Zn. Moreover, the EC50 values of BBF were higher than those of BL in Cd and Pb, similar to that of Cu and lower than that of BL in Zn. The effects of heavy metals on RM and OT on F1 were also concentration-dependent, showing in Figs. 2 and 3 and Table 1. Significant inhibition effects (p o0.05) were found for all the tested heavy metals as compared to the concurrent control. However, similar with P0, the observed inhibition effects on RM and OT were no more than 50 percent (POC values all higher than 50 percent), therefore could not generate any EC50 values (Table 1). Comparing the inhibition effects on all the tested indicators between P0 and F1, more severe effects were observed in F1, except for the inhibition effects of Cd exposure on OT at high concentrations (Fig. 3).

Based on the above results, the heavy metals indeed caused transgenerational effects in the progeny. Different from earlier reports (Wang and Yang, 2007; Hu et al., 2008), the defects caused by heavy metal exposure were more severe in progeny than those in the exposed parent, without any recovery or rescue. Moreover, EC50 values in the progeny at environmental concentrations were also calculated in the present study. Varied from earlier studies using L4 larva stage (Wang et al., 2007a; Wang and Yang, 2007), we employed L3 larva stage, with an exposure time covering the formation of the sperm, ovum and the eggs of the progeny (Hill et al., 2006), and exhibited more severe transgenerational effects. The important role of the prenatal period was also demonstrated in earlier studies on the transgenerational effects of SSRIs, BPA and perfluorinated chemicals (Salian et al., 2011; Grzeskowiak et al., 2012; Okada et al., 2012). To explain the transgenerational effects, earlier studies on the distribution of toxicants in C. elegans might provide some insights. The transition of toxicants from the alimentary system to the reproductive system was demonstrated on quantum dots (Qu et al., 2011), graphite nanoplatelets (Zanni et al., 2012), and silica-nanoparticles (Pluskota et al., 2009). Evidences on the transgenerational transfer of fluorescent nanodiamonds from the parent to the embryos and eventually into the hatched larvae in the next generations were also reported (Mohan et al., 2010). Since the metal ions have smaller sizes than the nanoparticles, the metals might have greater transgenerational abilities, which can be demonstrated by the capacities of heavy metals to cross the human placenta (Soghoian et al., 2006). Other potential explanations for the transgenerational effects of heavy metals might be related to the genetic or epigenetic reasons, which need further studies. 3.3. The ratio of EC50,

parent/EC50, progeny

The ratio of EC50, P0/EC50, F1 (Table 1) was used to indicate the difference of toxicities between two generations, where the value greater than 1 means greater toxicities on the progeny than on the parent. The higher the ratio of EC50, P0/EC50, F1 is, the more differences between two generations are, and therefore the greater the transgenerational effects will be. The determined ratios of EC50, P0/EC50, F1 were generally less than 10 for these tested metals, but the ratio reached as high as 35.6 for the effects of Cd on BL. Based on the absolute EC50 values for the effects on the parent or the progeny, cadmium was not the most toxic metal for either generation, however, its EC50 difference between P0 and F1 was the most significant, indicating that cadmium has greater

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Z. Yu et al. / Ecotoxicology and Environmental Safety 88 (2013) 178–184

Cd *

100

Pb

Cu

*

* *

*#

Percentage of control

*#+

*# *#

*

*#+*#

80

Zn * * *#

* *#

*# *#+ *+

*+ *#

*+

*#

*#

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60

*# *#+ *#+*# *

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20

Co nt ro 3. l 8E -0 2 3. 8E -0 3. 1 8E +0 0 3. 8E + 3. 01 8E +0 2

Co nt ro 4. l 7E -0 2 4. 7E -0 4. 1 7E + 4. 00 7E +0 1 4. 7E +0 2 Co nt ro 4. l 8E -0 3 4. 8E -0 4. 2 8E -0 4. 1 8E +0 0 4. 8E +0 1

Co nt ro 2. l 2E -0 2. 2 2E -0 2. 1 2E + 2. 0 0 2E +0 1 2. 2E +0 2

0

Fig. 2. The effects of cadmium (Cd), copper (Cu), lead (Pb) and zinc (Zn) on the reversal movement of the nematode parent (P0, blank) after prenatal exposure and the indirectly exposed nematode progeny (F1, shade). *: significantly different from the control, po 0.05; ]: significantly different from the lower concentration, p o0.05; þ: significantly different effects in progeny than in parent, p o0.05.

Cd

Percentage of control

100

*

Cu

* ** *

80

*#+ * *#

Pb *

*

*

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*

**

*

**

*+ *+

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60

*

Zn

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20

0

Fig. 3. The effects of cadmium (Cd), copper (Cu), lead (Pb) and zinc (Zn) on the Omega turn of the nematode parent (P0, blank) after prenatal exposure and the indirectly exposed nematode progeny (F1, shade). *: significantly different from the control, p o0.05; ]: significantly different from the lower concentration, po 0.05; þ: significantly different effects in progeny than in parent, po 0.05.

Cd 100

Cu

Percentage of control

80

*+ *#

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40

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Zn

Pb *

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20

Co nt ro 3. l 8E -0 2 3. 8E -0 1 3. 8E +0 3. 0 8E + 3. 01 8E +0 2

Co nt ro l 8E -0 3 4. 8E -0 4. 2 8E -0 4. 1 8E +0 0 4. 8E +0 1 4.

nt ro 4. l 7E -0 2 4. 7E -0 1 4. 7E + 4. 00 7E +0 1 4. 7E +0 2

Co

Co nt r 2. ol 2E -0 2. 2 2E -0 2. 1 2E +0 0 2. 2E +0 1 2. 2E +0 2

0

Fig. 4. The effects of cadmium (Cd), copper (Cu), lead (Pb) and zinc (Zn) on the body length of the nematode parent (P0, blank) after prenatal exposure and the indirectly exposed nematode progeny (F1, shade). *: significantly different from the control, p o0.05; ]: significantly different from the lower concentration, po 0.05; þ: significantly different effects in progeny than in parent, po 0.05.

capacities to provoke transgenerational effects. Such contradictory findings suggested that EC50 values alone are not sufficient enough to interpret the toxicities of toxicants. In fact, such

insufficiency was also demonstrated in earlier reports. The toxicity sequences of metals were different when the comparison was based on LC50 ratios of day 1 to day 4, other than on LC50 values

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alone (Williams and Dusenbery, 1990). The difference was also found by the comparison between the results of LC50 (or EC50) and those of the ratios of LC50/EC50 (Dhawan et al., 2000). This finding suggests that to appropriately assess the risk of parent-progeny effects, additional parameters, e.g., EC50, parent/EC50, progeny, should be considered for more accurate evaluations.

4. Conclusion The results demonstrated the transgenerational effects of heavy metals on the test organism at environmentally realistic concentrations. L3 larva stage of C. elegans showed higher sensitivities than L4 larva stage in both parent and progeny, and therefore was more appropriate for transgenerational effect studies. Generally, the progeny suffered more severe effects than the parent, and the transgenerational effects implied that parental exposure can multiply the harmful effects of heavy metal pollution in following generations. Considering the transgenerational effect, the ratio of EC50, parent/EC50, progeny should be considered as an important parameters in toxicity studies, so as to provide more sound and accurate information in judging the environmental risks of toxicants.

Acknowledgments The authors are grateful for the financial supports by the Program of International Science and Technology Cooperation of PR China (2010DFA91800) and the Ph.D. Programs Foundation of Ministry of Education of China (20110072110021). References Anderson, G., Boyd, W., Williams, P., 2001. Assessment of sublethal endpoints for toxicity testing with the nematode Caenorhabditis elegans. Environ. Toxicol. Chem. 20, 833–838. Arambaˇsic´, M.B., Bjelic´, S., Subakov, G., 1995. Acute toxicity of heavy metals (copper, lead, zinc), phenol and sodium on Allium Cepa L., Lepidium sativum L., and Daphnia magna St.: comparative investigations and the practical applications. Water Res. 29, 497–503. Blaxter, M., Denver, D.R., 2012. The worm in the world and the world in the worm. BMC Biol., 10. 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. Cain, D., Croteau, M.N., Luoma, S., 2011. Bioaccumulation dynamics and exposure routes of Cd and Cu among species of aquatic mayflies. Environ. Toxicol. Chem. 30, 2532–2541. Darr, D., Fridovich, I., 1995. Adaptation to oxidative stress in young, but not in mature or old, Caenorhabditis elegans. Free Radical Bio. Med. 18, 195–201. de Bono, M., Maricq, A.V., 2005. Neuronal substrates of complex behaviors in C. elegans. Annu. Rev. Neurosci. 28, 451–501. 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. Emmons, S., Klass, M., Hirsch, D., 1979. Analysis of the constancy of DNA sequences during development and evolution of the nematode Caenorhabditis elegans. Proc. Nat. Acad. Sci. U.S.A. 76, 1333–1337. Grzeskowiak, L.E., Gilbert, A.L., Morrison, J.L., 2012. Long term impact of prenatal exposure to SSRIs on growth and body weight in childhood: evidence from animal and human studies. Reprod. Toxicol. 34, 101–109. Guo, Y.L., Yang, Y.C., Wang, D.Y., 2009. Induction of reproductive deficits in nematode Caenorhabditis elegans exposed to metals at different developmental stages. Reprod. Toxicol. 28, 90–95. Hill, R., Egydio de Carvalho, C., Salogiannis, J., Schlager, B., Pilgrim, D., Haag, E., 2006. Genetic flexibility in the convergent evolution of hermaphroditism in Caenorhabditis Nematodes. Dev. Cell 10, 531–538. Hu, Y.O., Wang, Y., Ye, B.P., Wang, D.Y., 2008. Phenotypic and behavioral defects induced by iron exposure can be transferred to progeny in Caenorhabditis elegans. Biomed. Environ. Sci. 21, 467–473. IARC, 1997. Cadmium and cadmium compounds, IARC monographs on the evaluation of carcinogenic risks to humans. International Agency for Research on Cancer, 58. World Health Organization 119.

183

Leung, M.C.K., Williams, P.L., Benedetto, A., Au, C., Helmcke, K.J., Aschner, M., Meyer, J.N., 2008. Caenorhabditis elegans: an emerging model in biomedical and environmental toxicology. Toxicol. Sci. 106, 5–28. Loria, P.M., Hodgkin, J., Hobert, O.A., 2004. Conserved postsynaptic transmembrane protein affecting neuromuscular signaling in Caenorhabditis elegans. J. Neurosci. 24, 2191–2201. Mohan, N., Chen, C.-S., Hsieh, H.-H., Wu, Y.-C., Chang, H.-C., 2010. In vivo imaging and toxicity sssessments of fluorescent nanodiamonds in Caenorhabditis elegans. Nano Lett. 10, 3692–3699. Okada, E., Sasaki, S., Saijo, Y., Washino, N., Miyashita, C., Kobayashi, S., Konishi, K., Ito, Y.M., Ito, R., Nakata, A., Iwasaki, Y., Saito, K., Nakazawa, H., Kishi, R., 2012. Prenatal exposure to perfluorinated chemicals and relationship with allergies and infectious diseases in infants. Environ. Res. 112, 118–125. ¨ Polkki, M., Kangassalo, K., Rantala, M.J., 2012. Transgenerational effects of heavy metal pollution on immune defense of the blow fly Protophormia terraenovae. PLoS One 7, e38832, doi:38810.31371/journal.pone.0038832. Pinho, G.L.L., Bianchini, A., 2010. Acute copper toxicity in the euryhaline copepod Acartia tonsa implications for the development of an estuarine and marine biotic ligand model. Environ. Toxicol. Chem. 29, 1834–1840. Pluskota, A., Horzowski, E., Bossinger, O., von Mikecz, A., 2009. Caenorhabditis elegans nanoparticle-bio-interactions become transparent: silica-nanoparticles induce reproductive senescence. PLoS One 4, e6622. Qu, Y., Li, W., Zhou, Y.L., Liu, X.F., Zhang, L.L., Wang, L.M., Li, Y.F., Iida, A., Tang, Z.Y., Zhao, Y.L., Chai, Z.F., Chen, C.Y., 2011. Full assessment of fate and physiological behavior of quantum dots utilizing Caenorhabditis elegans as a model organism. Nano Lett. 11, 3174–3183. Reinke, S.N., Hu, X., Sykes, B.D., Lemire, B.D., 2010. Caenorhabditis elegans diet significantly affects metabolic profile, mitochondrial DNA levels, lifespan and brood size. Mol. Gen. Metab. 100, 274–282. Salian, S., Doshi, T., Vanage, G., 2011. Perinatal exposure of rats to Bisphenol a affects fertility of male offspring-An overview. Reprod. Toxicol. 31, 359–362. Santos, W.P.C.D., Gramacho, D.R., Teixeira, A.P., Costa, A.C.S., Korn, M. d.G.A., 2008. Use of Doehlert design for optimizing the digestion of beans for multi-element determination by inductively coupled plasma optical emission spectrometry. J. Braz. Chem. Soc. 19 (1–10). Soghoian, S., Sinert, R., Ferner, D.J., 2006. Toxicities, Heavy Metals. Source: /http:// www.emedicine.com/emerg/topic237.htm#section~introductionS. Suresh, G., Sutharsan, P., Ramasamy, V., Venkatachalapathy, R., 2012. Assessment of spatial distribution and potential ecological risk of the heavy metals in relation to granulometric contents of Veeranam lake sediments, India. Ecotoxicol. Environ. Saf. 84, 117–124. Swanson, J.M., Entringer, S., Buss, C., Wadhwa, P.D., 2009. Developmental origins of health and disease: environmental exposures. Semin. Reprod. Med. 27, 391–402. Van Gilst, M.R., Hadjivassiliou, H., Yamamoto, K.R., 2005. A Caenorhabditis elegans nutrient response system partially dependent on nuclear receptor NHR-49. Proc. Nat. Acad. Sci. U.S.A. 102 (13496–13501). Wang, C., Liu, S.L., Zhao, Q.H., Deng, L., Dong, S.K., 2012. Spatial variation and contamination assessment of heavy metals in sediments in the Manwan Reservoir, Lancang River. Ecotoxicol. Environ. Saf. 82, 32–39. Wang, D., Shen, L., Wang, Y., 2007a. The phenotypic and behavioral defects can be transferred from zinc-exposed nematodes to their progeny. Environ. Toxicol. Pharmacol. 24, 223–230. Wang, D., Wang, Y., 2008. Nickel sulfate induces numerous defects in Caenorhabditis elegans that can also be transferred to progeny. Environ. Pollut. 151, 585–592. Wang, D.Y., Yang, P., 2007. Multi-biological defects caused by lead exposure exhibit transferable properties from exposed parents to their progeny in Caenorhabditis elegans. J. Environ. Sci. 19, 1367–1372. Wang, Y., Xie, W., Wang, D.Y., 2007b. Transferable properties of multi-biological toxicity caused by cobalt exposure in Caenorhabditis elegans. Environ. Toxicol. Chem. 26, 2405–2412. Williams, P.L., Dusenbery, D.B., 1990. Aquatic toxicity testing using the nematode, Caenorhabditis elegans. Environ. Toxicol. Chem. 9, 1285–1290. Wu, Q.L., Nouara, A., Li, Y.P., Zhang, M., Wang, W., Tang, M., Ye, B.P., Ding, J.D., Wang, D.Y., 2012a. Comparison of toxicities from three metal oxide nanoparticles at environmental relevant concentrations in nematode Caenorhabditis elegans. Chemosphere 10.1016-j.chemosphere.2012.1009.1019. Wu, Q.L., Wang, W., Li, Y.X., Li, Y.P., Ye, B.P., Tang, M., Wang, D.Y., 2012b. Small sizes of TiO2-NPs exhibit adverse effects at predicted environmental relevant concentrations on nematodes in a modified chronic toxicity assay system. J. Hazard. Mater. 10.1016-j.jhazmat.2012.1010.1013. Yi, Y., Yang, Z., Zhang, S., 2011. Ecological risk assessment of heavy metals in sediment and human health risk assessment of heavy metals in fishes in the middle and lower reaches of the Yangtze River basin. Environ. Pollut. 159, 2575–2585. Yu, T., Zhang, Y., Hu, X.N., Meng, W., 2012a. Distribution and bioaccumulation of heavy metals in aquatic organisms of different trophic levels and potential health risk assessment from Taihu lake, China. Ecotoxicol. Environ. Saf. 81, 55–64. Yu, Z.Y., Jiang, L., Yin, D.Q., 2011. Behavior toxicity to Caenorhabditis elegans transferred to the progeny after exposure to sulfamethoxazole at environmentally relevant concentration. J. Environ. Sci.—China 23, 294–300. Yu, Z.Y., Zhang, J., Yin, D.Q., 2012b. Toxic and recovery effects of copper on Caenorhabditis elegans by various food-borne and water-borne pathways. Chemosphere 87, 1361–1367.

184

Z. Yu et al. / Ecotoxicology and Environmental Safety 88 (2013) 178–184

Zanni, E., De Bellis, G., Bracciale, M.P., Broggi, A., Santarelli, M.L., Sarto, M.S., Palleschi, C., Uccelletti, D., 2012. Graphite nanoplatelets and Caenorhabditis elegans: insights from an in vivo model. Nano Lett. 12, 2740–2744. Zeng, H.C., Li, Y.Y., Zhang, L., Wang, Y.J., Chen, J., Xia, W., Lin, Y., Wei, J., Lv, Z.Q., Li, M., Xu, S.Q., 2011. Prenatal exposure to perfluorooctanesulfonate in rat

resulted in long-lasting changes of expression of synapsins and synaptophysin. Synapse 65, 225–233. Zheng, N., Wang, Q.C., Liang, Z.Z., Zheng, D.M., 2008. Characterization of heavy metal concentrations in the sediments of three freshwater rivers in Huludao City, Northeast China. Environ. Pollut. 154, 135–142.