Arch Environ Contam Toxicol DOI 10.1007/s00244-014-0125-4

Growth Inhibition and Altered Gene Transcript Levels in Earthworms (Eisenia fetida) Exposed to 2,20 ,4,40 -Tetrabromodiphenyl Ether Xiang-bo Xu • Ya-juan Shi • Yong-long Lu Xiao-qi Zheng • R. J. Ritchie



Received: 2 September 2014 / Accepted: 29 December 2014 Ó Springer Science+Business Media New York 2015

Abstract The toxic effects of the ubiquitous pollutant 2,20 ,4,40 -tetrabromodiphenyl ether (BDE-47) on the earthworm Eisenia fetida were assessed by determining growthinhibition and gene transcript levels of superoxide dismutase (SOD), catalase (CAT), glutathione transferase (GST), and transcriptional changes of the stress-response gene (heatshock protein 70 [Hsp70]). Somatic growth and growthinhibition rates in all BDE-47-treated groups were significantly different from those of the controls. The SOD gene transcripts were upregulated at all exposure doses and reached the maximum at the concentration of 400 mg/kg dry weight (dw) (3.84-fold, P \ 0.01), which protected earthworms from oxidative stresses. However, downregulation of CAT and Hsp70 was present in all exposure doses and reached to the minimum at concentrations of 400 mg/kg dw

X. Xu  Y. Shi (&)  Y. Lu  X. Zheng State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China e-mail: [email protected] X. Xu  X. Zheng University of Chinese Academy of Sciences, Beijing 100049, China X. Xu Beijing Municipal Environmental Monitoring Center, Beijing 100048, China X. Zheng National Center for Climate Change Strategy and International Cooperation, Beijing 100038, China R. J. Ritchie Tropical Environmental Plant Biology Unit, Faculty of Technology and Environment, Prince of Songkla University – Phuket, Kathu, Phuket 83120, Thailand

(0.07-fold, P \ 0.01 and 0.06-fold, P \ 0.01, respectively). Upregulation of GST gene transcript level presented significant changes at concentrations of 10 (2.69-fold, P \ 0.05) and 100 mg/kg dw (2.55-fold, P \ 0.05). SOD maintained a dynamic balance to upregulate SOD expression to eliminate superoxide radicals in all dosage treatments, but downregulation of CAT decreased the ability to eliminate hydrogen peroxide. These changes could result in biochemical and physiological disturbances in earthworms.

Polybrominated diphenyl ethers (PBDEs) have been widely used as flame retardants. 2,20 ,4,40 -Tetrabromodiphenyl ether (BDE-47) is one of the most prevalent congeners of PBDEs and a ubiquitous pollutant found in many media such as water, air, soil, serum, milk, fish, and harbor seal blubber (Christensen et al. 2002; She et al. 2002; Jaward et al. 2004; Bi et al. 2006; Inoue et al. 2006; Duan et al. 2010). A variety of reports have shown that BDE-47 has reproductive toxicity and endocrine-disrupting effects to model indicator species such as mussels (Gastropoda), mice, and zebrafish (Vertebrata). Damaging effects on ovarian follicles and ovocytes were observed in BDE-47exposed female mussels Mytilus edulis (Aarab et al. 2006). Low doses of BDE-47 on the female reproductive system of mouse Leydig tumor cells leading to a significant decrease in ovarian weight has been evaluated (Talsness et al. 2008). BDE-47 exposure significantly affected spontaneous movement and decreased touch response and free swimming speed during early zebrafish developmental stages (Usenko et al. 2011; Chen et al. 2012). Earthworms are widespread in soils and are often used as model species to assess the toxic effects of chemicals on soil ecosystems. Earthworm (E. fetida) is an attractive

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biological model because it is available commercially, easy to handle, susceptible to chemicals, and can be bred readily in a wide range of organic waste materials (Organisation for Economic Co-operation and Development (OECD) 1984). Mortality and growth inhibition of earthworms (E. fetida) have been verified as effective indicators in a variety of reports (Shi et al. 2007; Wu et al. 2011; Xie et al. 2011). As a sensitive indicator, gene transcript level has been used in assessing toxic effects of toxicants to earthworms (E. fetida) (Asensio et al. 2007; Brulle et al. 2007, 2008; Zheng et al. 2008). Antioxidant enzymes, such as SOD, CAT, and glutathione transferase (GST) can protect organisms from oxidative damage by removing reactive oxygen species (ROS). ROS not only regulate the activity of pre-existing proteins or enzymes, they are also responsible for inducing the expression of many genes (Chen et al. 2011a). Heat-shock protein 70 (Hsp70) also plays an important protective role in cellular stress responses and had been used in many toxicity tests (Homa et al. 2005, 2007; Chen et al. 2011a). Biochemical responses of earthworms (E. fetida) to some congeners of PBDEs have been reported. Antioxidant responses induced by decabromodiphenyl ether (BDE-209) have been reported in earthworms (E. fetida) (Xie et al. 2011). Activities of superoxide dismutase (SOD), CAT, and peroxidase were measured to study the toxic effects of 2,30 ,40 ,6-tetrabromodiphenyl ether (BDE-71) in earthworms (E. fetida) (Zhu et al. 2010). Metabolic and proteomic responses of E. fetida showed that BDE-47 mainly caused disturbances in osmotic regulation and energy metabolism and induced cell apoptosis, oxidative stress, and disturbance in protein biosynthesis (Ji et al. 2013). However, genotoxicity of antioxidant and cellular stress responses of earthworms to BDE-47 are rarely reported and warrant more thorough investigation. In this study, acute tests (14-day) were performed to indicate the toxic effects of BDE-47 on earthworms (E. fetida) by detecting growth-inhibition and gene transcript levels of SOD, CAT, GST, and Hsp70. The study may provide a scientific database for soil ecological risk assessment and clarify the toxicity mechanism of BDE-47 to the earthworm (E. fetida), which has a keystone role in composting.

Materials and Methods Earthworms and Chemicals The earthworms (E. fetida) were purchased from Earthworm Runfeng Company in Beijing, China. Before starting the test, adult earthworms (0.35–0.45 g) with well-developed clitellum were chosen to acclimatize in artificial soils (described later in the text) for 24 h. Before the initiation of

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the exposure test, earthworms were rinsed in deionized water and moved to Petri dishes on moist filter paper (in the dark at 20 ± 1 °C for 24 h) to void their gut contents. BDE-47 (CAS no. 5436-43-1, 99.5 % purity) was purchased from Chem Service (West Chester, Pennsylvania, USA). N-hexane (analytical grade) and all of the other chemicals and reagents (analytical grade) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All aqueous solutions were prepared by using reagent water from a Milli-Q Gradient system (Millipore Company, Bedford, Massachusetts, USA). Acute Toxicological Tests The earthworm cultivation method followed the guidelines of the OECD method (OECD 1984). The artificial soils consisted of 70 % quartz sand, 20 % kaolin clay, and 10 % sphagnum peat, and the moisture content was adjusted to approximately 35 % by adding deionized water. The pH is adjusted to 6.0 ± 0.5 with calcium carbonate. BDE-47 was spiked with n-hexane (25 ml) as carrier and thoroughly mixed into the artificial soils at concentrations of 0, 10, 50, 100, 200, and 400 mg/kg dry soil (four containers per group; translucent beakers used as containers). Artificial soils, 750 g wet weight, were added to each container. Controls were four containers without BDE-47. Before adding the earthworms, every container was placed in an exhaust hood for 2–4 days to ensure that the n-hexane had completely evaporated. Ten earthworms (0.35–0.45 g each) were then added to each container. The containers were kept in an incubation chamber (20 ± 1 °C) at 83 ± 3 % relative humidity with continuous illumination at 400–800 lux throughout the test period. During the period of exposure, earthworms were taken out of the containers, cleaned with deionized water, and weighed on days 4, 7, 10, and 14. After exposure for 14 days, three earthworms were selected from each container randomly to be used to determine gene transcript levels.

Growth-Inhibition and Somatic Growth Rates of Earthworms Earthworms of all containers were weighed on days 4, 7, 10, and 14, the growth-inhibition rate and somatic growth rate formulas are as follows: GIRn ¼

W0  Wj  100% W0

SGRn ¼

ln Wj  ln Wi  100% , tj  ti

where GIRn is the growth-inhibition rate for dose group n; SGRn is the somatic growth rate for dose group n from day

Arch Environ Contam Toxicol Table 1 Sequence of primers used for RT-qPCR Gene

Accession no.

Forward primer

Reverse primer

b-actin

GU177854

50 -TCCATCGTCCACAGAAAG-30 0

SOD

GU177856

5 -TGCTCACTTCAACCCATTT-3

CAT

GU177857

50 -CATTGCGGATGGAAACTA-3

GST Hsp70

HQ693699 GU177858

50 -AAATGTCCTCCGCAAGCT-30 0

50 -TTGGCAACACCACTTTCA-30 50 -CCAAGGACAACAACCTGCTC-30

0

5 -ATGCCATTCTGCGCTACGTTGC-3 0

5 -CCAAGGACAACAACCTGCTC-3

i to j; W0 is the weight on day 0; Wi is the weight on day i and Wj is the weight after j days of exposure (Shi et al. 2007; OECD 2006).

0

0

50 -TCCGGCGCCTCCTTGATTTTC-30 50 -CGGCGTTCTTCACCATTC-30

target gene (Tg), and b-actin gene(act) (Pfaffl 2001; Brulle et al. 2006). Statistical Analysis

Gene Transcript Levels Earthworms were selected from each container randomly, cleaned with deionized water, left in Petri dishes with moist filter paper to void their gut contents for 24 h, and then ground immediately in liquid nitrogen. Total RNA was isolated using the TRIzol Reagent (Invitrogen, USA), according to the manufacturer’s instructions. RNA purity and integrity were checked by agarose gel electrophoresis (2 %) to ensure that absorbance ratios (A260/280) were between 1.8 and 2.0. First-strand cDNA synthesis was performed using a superscriptTM II reverse transcriptase (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions (Chen et al. 2011b). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) of the selected genes (b-actin, SOD, CAT, GST, and Hsp70) were performed using the RealTime Polymerase Chain Reaction kit (TaKaRa, Tokyo, Japan). Each amplification reaction mixture (20 ll) contained 2.0 ll cDNA template, 0.4 ll each of forward and reverse primer (concentration 10 lM), 6.8 ll ddH2O, 10 ll SYBR Premix Ex Taq (29), and 0.4 ll ROX Reference Dye(509)*2. The following RT-qPCR reactions were performed with the 7300 Real-Time PCR system (ABI, Foster, CA, USA): initial denaturation for 10 min at 95 °C was followed by 40 cycles of 15 s at 95 °C, 15 s at 55 °C, and 45 s at 72 °C, and dissociation by 1 cycle of 15 s at 95 °C, 30 s at 55 °C, and 15 s at 95 °C. Sequence of primers used for RT-qPCR are listed in Table 1. Primers of b-actin, SOD, CAT, and Hsp70 used for earthworms (E. fetida) were selected from references (Brulle et al. 2006; Chen et al. 2011b).The primers of GST were designed based on known sequences (HQ693699). RT-PCR efficiencies (E) were calculated from the given slope of the standard curve according to the equation E = 10(-1/slope) - 1. They ranged from 0.98 to 1 (Pearson correlation coefficient r2 [ 0.99), and a strong linearity was observed. The gene transcript levels (R) were calculated according to the formula R = (ETg)CPTg/(Eact)CPact,

All statistical analyses were performed using SPSS 18.0 software (SPSS, Chicago, Illinois, USA), and all charts were designed using Origin 8.6 software (Origin, Northampton, MA,USA). Within meeting the conditions of the normality assumption and the homogeneity of variances, which were tested, respectively, by Shapiro–Wilk test and Levene’s test, and data for growth-inhibition and gene transcript levels were each subjected to one-way analysis of variance (ANOVA) using Fisher’s least significant difference test for contrast of the differences among treatment means (Shi et al. 2007). Logarithmic transformation was applied to the dependent variables in gene transcript level analysis. Pearson correlation was used in correlation analysis. A level of 0.05 was used in all procedures (equivalent to 95 % confidence), and data are presented as mean ± SD.

Results Growth Inhibition Somatic growth rates of earthworms at a specific period are shown in Fig. 1a. Negative somatic growth rates portended growth inhibition of earthworms, and absolute values of all groups were greater with the increase of BDE-47 concentrations. The minimum value of somatic growth rate appeared at the concentration of 400 mg/kg dw (-4.26 %, P \ 0.001) during the period of 7–10 days. Somatic growth rates of earthworms showed growth inhibition of BDE-47 to earthworms, which was confirmed by the growth-inhibition rates. The growth-inhibition rates for BDE-47 after exposures of 4, 7, 10, and 14 days are shown in Fig. 1b. Concentration-related growth inhibition in earthworms from acute exposure to BDE-47 was significant. Due to the decrease of soil nutrients, the growth inhibition of the control group increased with increased exposure time, but it was lower

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than the growth-inhibition rates of the BDE-47 groups. After 4, 7, 10, and 14 days of exposure to BDE-47, the growth-inhibition rates of earthworms treated with BDE-47 at concentrations of 200 and 400 mg/kg dw were significantly greater than those for worms treated at other concentrations. The decreases in weight after 4-, 7-, 10-, and 14-day exposures to BDE-47 were found statistically to be dose-dependent (Pearson correlation r = 0.884, P \ 0.01; r = 0.958, P \ 0.01; r = 0.959, P \ 0.01 and r = 0.971, P \ 0.01, respectively). Furthermore, the growth-inhibition rates in the treatment group of 400 mg/kg dw reached a maximum of 36.4 % at day 14 of the experiment (P \ 0.01).

Gene Transcript Levels Transcript levels of four target genes (SOD, CAT, GST, and Hsp70) showed significant variations between dose groups and the control throughout the 14-day acute tests (Fig. 2). With increased concentrations, significant (P \ 0.05) upregulation of SOD transcript level was observed in earthworms, and the highest level of SOD gene transcripts appeared at the concentration of 400 mg/kg dw (3.84-fold, P \ 0.01). Earthworms exposed to BDE-47 expressed a significant (P \ 0.05) downregulation of CAT (100, 200, and 400 mg/kg dw) and Hsp70 (100, 200 and 400 mg/kg dw) gene transcript levels. GST gene transcript levels reached the maximum value at a dosage of 10 mg/kg dw (2.69-fold, P \ 0.05); thereafter, they decreased to the control level except at the concentration of 200 mg/kg dw (P \ 0.05). Multiple relationships of all gene transcript levels in dose groups compared with the control level and with their statistical differences are listed in Table 2.

Discussion In this study, we focused on the toxic effects of BDE-47 for a 14-day exposure on the earthworm (E. fetida) using a laboratory-spiked soil method, and the indicators were growth inhibition and gene transcript levels. Biomass changes could be a good indicator of chemical stress and link chemical effects to energy dynamics and, ultimately, to growth inhibition (Shi et al. 2007; Wu et al. 2012). Previous reports indicated that some monomers of PBDEs significantly inhibited the growth of the earthworm (E. fetida). BDE-71 (1,000 mg/kg dw) had limited effects on mortality (3–4 %), but it had significant effects on

Fig. 1 Acute growth responses of earthworms (E. fetida) exposed to BDE-47 for 4, 7, 10, and 14 days in an artificial-soils test. BDE-47 significantly inhibited growth of the worms compared with that of the controls and was dosage-dependent. Data are presented as mean ± SD (n = 4). Significant difference from control values at *P \ 0.05, **P \ 0.01, or ***P \ 0.001

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Fig. 2 Changes in target genes in earthworm (E. fetida) tissues with different BDE-47 treatments after 14 days exposure. Significant difference from control values at *P \ 0.05 or **P \ 0.01

Arch Environ Contam Toxicol Table 2 Multiple relationships and P values for SOD, CAT, GST, and Hsp70 in earthworms exposed to BDE-47 BDE-47 Concentrations (mg/kg dw)

Multiple relationships and P values SOD

CAT

GST

Hsp70

10

1.32

1.18

2.69

1.08

NS

NS

0.012

NS

50

1.45

0.21

1.04

0.23

NS

NS

NS

NS

2.12

0.17

2.55

0.18

NS

0.004

0.043

0.022

2.22

0.13

1.19

0.15

0.049

0.002

NS

0.017

3.84

0.07

0.61

0.06

0.002

0.001

NS

0.008

100 200 400

NS = P [ 0.05 Significant difference compared with controls (P \ 0.05)

growth (GIR = 25 %) on E. fetida after 14 days of exposure (Zhu et al. 2009); however, BDE-209 (1,000 mg/kg dw) had no significant effects on the growth of earthworms (E. fetida) after a 28-day exposure (Xie et al. 2013). In this study, the slight decrease of earthworm weight in the control treatment suggested that the soil nutrients were insufficient to allow for additional growth. Despite this, the growth-inhibition and somatic growth rates of all BDE-47 dose groups were significantly different than those of controls (Fig. 1). The growth-inhibition rates in the treatment group of 400 mg/kg dw BDE-47 reached a maximum of 36.4 % after 14-day exposure, but no deaths appeared to occur in all treatment groups. Ribeiro et al. (2001), working on isopods (Porcellio dilatatus), found that feeding inhibition decreased consumption rates and regulated the intake of the pesticide, thus resulting in decreased growth rate. This strategy of decreasing food intake to avoid toxins was introduced to explain the increasing growth inhibition of the earthworms in similar ecotoxicology tests (Bibicˇ et al. 2007; Mosleh et al. 2003; Shi et al. 2007). It appears the same strategy was used to avoid the toxic BDE-47 by the earthworms in this experiment. With increased concentrations of BDE-47, the significant upregulation of SOD expression levels indicated that transcription of genes might be stimulated by oxidative stress. The results for SOD suggested that the earthworms (E. fetida) suffered more stress from BDE-47 at greater concentrations. Under stressful physiological conditions, SOD maintained a dynamic balance to upregulate SOD expression to meet the requirement of the organism to eliminate superoxide radicals. This finding was similar to results shown for tonalide (AHTN) at concentrations of 0–50 mg/kg dw (Chen et al. 2011a).

Downregulation of CAT gene transcript level appeared after a 14-day exposure to BDE-47 except at low concentrations of BDE-47 (10 mg/kg dw) compared with the control group. The reason for the change in CAT gene transcripts could be explained if they become inhibitors of CAT activity, when the superfluous ROS levels generated exceed the capacity of scavenging ROS by SOD and CAT (Lin et al. 2010). Jo et al. (2008) made a similar finding in oysters (Crassostrea gigas): CAT mRNA expression rapidly decreased, whereas SOD mRNA expression did not decrease after 7 days of exposure to Cd. This indicated that although sufficient SOD converted the superoxide radical to hydrogen peroxide (H2O2) and O2, a decrease in CAT led to a breakdown in the conversion of H2O2 into O2 and H2O, thus leading to a failure to completely remove H2O2, which is harmful to living tissue. Upregulation of GST gene transcripts presented significant changes at BDE-47 concentrations of 10 (2.69-fold, P \ 0.05) and 100 mg/kg dw (2.55-fold, P \ 0.05). The free radical reactions occurred rapidly. Ribera et al. (2001) found that benzo(a)pyrene free radical reactions in earthworms (E. fetida) were regulated mainly by glutathione metabolism even if GST activities were unaffected by the 14-day exposure to benzo(a)pyrene. We might infer that earthworms were mainly using glutathione metabolism to regulate the free radical reactions when exposed to the lowdose BDE-47; however, at greater dosages BDE-47 induced the organism to generate an excessive amount of radicals resulting in damage to certain tissues. Hsp70 gene transcript level was found to be upregulated in the Manila clam (Ruditapes philippinarum) after exposure to 5 lg/L BDE-47 for 15 days (Miao et al. 2014). In the present study, the Hsp70 gene transcript level showed regular changes, and significant downregulation appeared after exposure to BDE-47 (100 mg/kg dw, 0.18-fold, P \ 0.05; 200 mg/kg dw, 0.15-fold, P \ 0.05; 400 mg/kg dw, 0.06-fold, P \ 0.01). The regulation pattern of Hsp70 gene transcripts was complex when exposed to various chemicals. Effects of EDCs on Hsp70 expression were differential, e.g., 4-nonylphenol and 4-t-octylpheno caused downregulation, whereas bisphenol A caused upregulation in the copepod Tigriopus japonicus (Rhee et al. 2009). Similar findings that the Hsp70 gene transcript level showed downregulation when earthworms (E. fetida) were exposed to galaxolide and AHTN were found by Chen et al. (2011a). However, noticeable downregulation was observed after exposure to BDE-47, which is in good accordance with observations on CAT activity, suggesting that the downregulation of Hsp70 gene transcripts might be influenced by H2O2. Further studies are required to study the possible mechanism of the regulation of Hsp70 gene transcripts, especially in response to BDE-47.

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Compared with the controls, the decrease of earthworm weight suggested that the toxic effects of BDE-47 could be detected using easily measureable external traits. Through analysis of gene expression, SOD maintained a dynamic balance to upregulate SOD expression to eliminate superoxide radical in all of the dose groups in the present study, GST could upregulate the free radical reactions even in the lowest-dose group. However, downregulation of CAT broke down the dynamic balance of elimination of H2O2, and then earthworm body might be hurt by the accumulation of H2O2. Growth inhibition of all BDE-47 dose groups was significantly greater than that of the controls, and growth inhibition was a sensitive biomarker of toxicity of BDE-47 in the earthworm (E. fetida). The sensitive alteration in gene transcript levels of SOD, CAT, GST, and Hsp70 showed that emerging oxidative stress occurred in the body of earthworms (E. fetida) and might provide a potential indicator to monitor the toxic effects of BDE-47 on earthworms (E. fetida) in field situations. Significant regular alterations of the Hsp70 gene transcripts presented here will provide insight in understanding the molecular mechanism in earthworms (E. fetida). Acknowledgments The authors are grateful for the support provided by the National Natural Science Foundation of China (Grant No. 41272487), the International S&T Cooperation Program of China (Grant No. 2012DFA91150), and the Key Research Program of the Chinese Academy of Sciences (Grant No. KZZD-EW-TZ-12).

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Growth inhibition and altered gene transcript levels in earthworms (Eisenia fetida) exposed to 2,2',4,4'-tetrabromodiphenyl ether.

The toxic effects of the ubiquitous pollutant 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) on the earthworm Eisenia fetida were assessed by determining...
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