Metallothionein-like proteins turnover, Cd and Zn biokinetics in the dietary Cd-exposed scallop Chlamys nobilis

Aquatic Toxicology 105 (2011) 361–368

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Metallothionein-like proteins turnover, Cd and Zn biokinetics in the dietary Cd-exposed scallop Chlamys nobilis Fengjie Liu, Wen-Xiong Wang ∗ Division of Life Science, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong

a r t i c l e

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Article history: Received 27 April 2011 Received in revised form 11 July 2011 Accepted 12 July 2011 Keywords: Chlamys nobilis Dietary metals Metallothionein turnover Metal biokinetics Biomarker

a b s t r a c t In this study, we tested whether metallothionein-like proteins (MTLPs) affected the biokinetics of Cd and Zn in the scallops Chlamys nobilis following dietary Cd exposure. The scallops were fed Cd-contaminated diatoms for 8 or 40 days, and then the tissue Cd concentrations, MTLP turnover and their standing stocks, Cd and Zn kinetics were monitored. After 8 days of dietary Cd exposure (at 98 or 196 ␮g Cd g−1 ), their Cd levels were increased by 7.4–12.5 times, newly synthesized MLTPs by 1.7–2.1 times, and their MLTP stocks doubled. However, after 40 days of dietary Cd exposure (at 58 or 115 ␮g Cd g−1 ), MTLP synthesis and MTLP stocks did not change despite the fact that Cd bioaccumulation increased by 2.7–4.2 times. MTLPs played little role in the overall Cd and Zn uptake from either food or water, since enhanced MTLP induction did not improve the sequestration of newly incoming Cd or Zn. As MTLP-metal complexes degraded, the released Cd was immediately sequestered by newly synthesized MTLPs while most Zn was depurated. This may explain why scallops eliminated Cd more slowly than Zn. Induced proteins were shown to play a minor role in the detoxification of dietary Cd in C. nobilis, but the species can modify its Cd assimilation to reduce the impact of high dietary Cd levels. MTLPs are probably unsuitable as biomarkers for environmental monitoring involving C. nobilis. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Marine bivalves are among the most ecologically important aquatic invertebrates and commercially important seafood sources (Phillips, 1977; Luoma and Rainbow, 2008). Seafood consumption is a substantial source of non-essential element (e.g. cadmium, mercury, arsenic) exposure for some human populations (Falcó et al., 2006; Luoma and Rainbow, 2008). The concentrations of toxic metals in the environment and seafood are further elevated by anthropogenic activities (Blackmore, 1998; Luoma and Rainbow, 2008), resulting in increased exposure of humans to toxic metals. Some bivalves can accumulate high levels of Cd within their soft tissues even in a clean environment (Viarengo et al., 1993; Kruzynski, 2004; Pan and Wang, 2009b). For example, Cd in the whole wet tissue ranging from 4.3 to 9.0 ␮g g−1 has been reported in both wild and commercially captured scallops (Kruzynski, 2004). Up to 91 ␮g Cd g−1 has been found in the wet tissue of scallops from sites under little anthropogenic influence (Ashoka, 1999), and the natural oceanographic conditions of high background Cd concentration may contribute to the high body Cd in these animals. The Cd levels in these scallops are much higher than the current limits of 1 ␮g Cd g−1 wet weight (European Community), 2 ␮g Cd g−1 wet weight

∗ Corresponding author. E-mail address: [email protected] (W.-X. Wang). 0166-445X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2011.07.011

(Hong Kong, Australia and New Zealand), and 3.7 ␮g Cd g−1 wet weight (United States) (Kruzynski, 2004). To ensure seafood safety, significant efforts have been made to understand the underlying mechanisms of metal bioaccumulation in marine bivalves (Wang et al., 1996; Luoma and Rainbow, 2008). One biochemical component involved in metal-accumulation is metallothioneins or metallothionein-like proteins (MTLPs) (Roesijadi, 1992; Amiard et al., 2006). MTLPs are cysteine-rich metal-binding proteins, and for decades most studies have focused on their roles in the sequestration and detoxification of intracellular metals such as Cd, Zn and Hg (Nordberg, 1998; Klaassen et al., 1999; Nordberg, 2009). Recently, several studies on aquatic animals have highlighted the significance of MTLPs in metal biokinetics other than metal detoxification and homeostasis (Wang and Rainbow, 2010). For instance, individual clams Ruditapes philippinarum with high MTLP concentrations showed distinguishably high Cd assimilation efficiency (AE) from food (Shi and Wang, 2004). A positive relationship between Cd uptake from either water or food and induced MTLP concentration has also been found in the black seabream Acanthopagrus schlegeli (Long and Wang, 2005). The importance of MTLP for Cd elimination has been demonstrated in the clam Ruditapes decusstatus (Serafim and Bebianno, 2007), the scallop Chlamys nobilis (Liu and Wang, 2011), and the freshwater bivalve Corbicula fluminea (Baudrimont et al., 2003). However, there have been inconsistent findings about the physiological roles of MTLPs in metal biokinetics (Wang and Rainbow, 2005, 2010). This

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is mainly because MTLP turnover (synthesis and degradation) and its relationship with metal biokinetics remain largely untested in aquatic animals. MTLPs have frequently been used as biomarkers in environmental monitoring, as the proteins are readily induced by various toxicological stimuli (Pedersen et al., 1997; UNEP/RAMOGE, 1999; Mathiessen, 2000). However, the best MTLP concentration for pollution monitoring is still a subject of discussion (Mouneyrac et al., 2002; Amiard et al., 2006). In tests with several species from sites where high levels of bioavailable metals were present no MTLP induction was observed (Pedersen and Lundebye, 1996; Geffard et al., 2001; Berthet et al., 2003). Under laboratory conditions, MTLP induction is also sometimes limited, although strong enhancement of Cd concentration has been found in some marine animals (Amiard et al., 2006; Ng et al., 2008). One explanation could be that the physiological activity of MTLPs in metal-exposed organisms may be mainly reflected in increased turnover of MTLPs rather than any increase in their stocks (Mouneyrac et al., 2002; Amiard et al., 2006; Wang and Rainbow, 2010). A clear example comes from the nereid polychaete Perinereis aibuhitensis (Ng et al., 2008), where faster turnover but comparable MTLP concentrations have been observed in Cd-exposed animals. Our previous work has recently shown that the scallop C. nobilis with 6–24 ␮g Cd g−1 wet weight had both higher MTLP turnover and increased MTLP concentrations following waterborne Cd exposure (Liu and Wang, 2011). Taken together, these results highlight the importance of further studies of MTLP turnover to better understand Cd detoxification as well as MTLP’s utility as a biomarker of metal exposure. The present study was designed to answer two questions: Do MTLPs affect the biokinetics of Cd and Zn in the scallops following dietary Cd exposure, and if so, how? Is measuring MTLP turnover an alternative or complementary choice for monitoring metal pollution? Perhaps MTLPs can decrease intracellular concentrations of Cd and Zn in a labile form, thus facilitating their subsequent uptake. Or MTLP-bound Cd and Zn may have different susceptibilities to proteolysis resulting in differences in their elimination (Klaassen et al., 1999; Amiard et al., 2006). The scallop C. nobilis was chosen as a model organism for testing these hypotheses, since it is a commercially important subtropical marine bivalve with high body Cd and Zn concentrations (Kruzynski, 2004; Pan and Wang, 2008a, 2009b). Growing on the same clean coastal waters, the scallops attained much higher body Cd concentrations (2 ␮g Cd g−1 wet weight) than the other three bivalve species (0.3–0.5 ␮g Cd g−1 wet weight in the clams, the green mussels and the oysters) (Liu and Wang, unpublished data). Previous modeling efforts suggested that such high Cd accumulation by the scallops was primarily caused by the very high dietary assimilation efficiency and low efflux rate of Cd, even though the dissolved Cd concentration was low (Pan and Wang, 2008a).

2. Materials and methods 2.1. Animal collection The juvenile scallops C. nobilis were collected from Dapeng Bay in China’s Guangdong Province and transported to the laboratory immediately after collection. There were two batches with shell lengths ranging from 1.5 to 2.0 cm and 2.5 to 3.0 cm, and uniform scallops of similar shell size were selected for each batch of experiment. Throughout the period of laboratory acclimatization and experiments the C. nobilis were maintained in natural coastal seawater (pH 8.1) from Clear Water Bay, Hong Kong at 23 ± 1 ◦ C, and fed the diatom Thalassiosira weissflogii at the rate of about 1–2% of their body tissue dry weight per day.

2.2. Dietary Cd pre-exposure The scallops were pre-exposed to different concentrations of dietary Cd for either 8 or 40 days. The batch of smaller scallops was used in the 40-d exposure experiment because of the size limit of the ␥-detector available. No visible development of their gonads was observed during the entire experimental period. For each duration of exposure there were three treatments involving a control, medium or high concentration of Cd. Each day, the scallops were fed 1.5–3% of their body tissue with T. weissflogii on a dry weight basis with the planned Cd exposure over 8 h, and then placed in clean seawater for 16 h. The T. weissflogii diatoms were prepared by filtration and resuspension in f /2 medium minus EDTA, Zn and Cu. Following 2 days of growth, 25 or 250 ␮g Cd L−1 was added for another 3–4 days before the algae were harvested by centrifugation and presented to the scallops. These Cd concentrations were rather high, but were comparable to those documented in the seriously contaminated waters, such as in the Carnon River and the Restronguet Creek estuary (Luoma and Rainbow, 2008). The growth of the diatoms was significantly reduced under the Cd exposure. The resulting Cd concentrations are shown in Table 1. To minimize desorption of loosely absorbed Cd from the cells to the water during the feeding periods, resuspension and centrifugation were repeated 4–6 times. Preliminary experiment showed that less than 1% of Cd desorbed from the final centrifuged diatoms. For the medium Cd exposure treatment, the scallops were fed with a 1:1 (wet wt./wet wt.) mixture of clean and Cd-exposed diatoms, and its Cd concentration was calculated from those in the clean and the Cd-exposed diatoms. For the high Cd exposure treatment, the scallops were fed only the Cd-exposed diatoms. 2.3. Body Cd and MTLP concentrations Following the dietary Cd exposure, the soft tissues of the scallops were dissected, wet weighed, and dried at 80 ◦ C for 4 days. The dried tissue was microwave digested, and Cd concentrations were measured using a furnace atomic absorption spectrometer (PerkinElmer Analyst 800). The Cd recovery from a certified reference material (Standard Reference Material 2976, National Institute of Standards and Technology, USA) was more than 90%. The Cd concentration in the scallop was expressed as ␮g Cd g−1 wet weight. The MTLP concentrations in the whole wet tissues were determined by an 110m Ag saturation method (Scheuhammer and Cherian, 1991; Liu and Wang, 2011). A metallothionein standard (a mixture of MT-I and MT-II rabbit liver, Dalian Free Trade Zone United BoTai Bio-Tech) was used, with a recovery rate of >87% in each measurement. 2.4. Biokinetics of Cd, Zn and MTLP turnover Dissolved uptake, dietary assimilation and efflux of Cd and Zn, and MTLP synthesis and degradation were quantified using wellestablished radiotracer techniques (Ng et al., 2007; Liu and Wang, 2011). The influx rates of Cd and Zn were quantified in 0.22 ␮mfiltered seawater spiked with 109 Cd (1.04 and 1.07 ␮Ci L−1 for the 8 and 40-d Cd exposed scallops, respectively) and 65 Zn (1.05 and 1.20 ␮Ci L−1 for the 8 and 40-d Cd exposed scallops, respectively). The seawater was also spiked with 35 S (as cysteine, 3.16 and 2.50 ␮Ci L−1 for the 8 and 40-d Cd exposed scallops, respectively) to measure the de novo MTLP synthesis. One scallop was collected from each of 4 or 5 replicated beakers with radiolabeled seawater at 1, 4, 8 and 12 h. The whole wet tissue was dissected, wet weighed and ␥-counted. After the radioassay for Cd and Zn, the soft tissues were subjected to subcellular fractionation (Nash et al., 1981; Wallace et al., 2003; Giguère et al.,

2.3 ± 0.5a 17.1 ± 3.3b 28.7 ± 9.7c 3.3 ± 0.6a 9.0 ± 0.6b 13.7 ± 1.2c 0.46 ± 0.04 98.4 196.3 ± 51.4 0.46 ± 0.04 57.5 114.6 ± 30.3 8 8 8 40 40 40 D0.5 D98 D196 D0.5 D58 D115

Duration (d)

D: dietary exposure; numbers following ‘D’ indicate the Cd concentrations in the food in ␮g Cd g−1 . Data are mean ± standard deviation (n = 2 for dietary Cd; n = 5–6 for tissue Cd; n = 4–5 for MTLP; n = 4–5 for dissolved uptake rate; n = 7–8 for AE; n = 9–10 for ke ). Different letters indicate statistical significance at the 0.05 level of confidence.

0.040 ± 0.012a 0.039 ± 0.013a 0.030 ± 0.016a 71.3 ± 4.6a 76.1 ± 8.6a 76.1 ± 7.5a 23.2 ± 1.8a 28.4 ± 2.6b 23.0 ± 3.8a 91.8 ± 11.6a 96.8 ± 12.2a 95.7 ± 7.2a 0.007 ± 0.002a 0.009 ± 0.004a 0.006 ± 0.003a 90.0 ± 7.1a 77.8 ± 11.1b 75.1 ± 8.4b 4.4 ± 0.6a 5.5 ± 0.8a 5.0 ± 1.4a 1.44 ± 0.09a 1.49 ± 0.13a 1.38 ± 0.11a

ke (d

AE (%) Uptake (ng g−1 h−1 )

Cd biokinetics

MTLP (␮g g−1 ww.) ww.) Cd (␮g g Dietary Cd (␮g g−1 dw.)

3.0 ± 0.3a 6.4 ± 2.1b 5.7 ± 1.0b 33.5 ± 13.3a 26.7 ± 5.2a 39.2 ± 7.8a

ke (d− ) AE (%) Uptake (ng g−1 h−1 ) )

Zn biokinetics

−1 −1

Tissue concentrations Pre-exposure Treatment

Table 1 Cd concentrations, metallothionein-like protein (MTLP) concentrations, dissolved uptake rates, assimilation efficiencies (AEs), and efflux rate constants (ke ) for Cd and Zn observed in the scallop Chlamys nobilis exposed to dietary Cd for 8 or 40 days.

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2006). The operationally defined fraction of MTLP was obtained from the heat-stable cytosolic supernatant (Olafson et al., 1979; Bragigand and Berthet, 2003), and the fraction was not further purified because it contained only a small number of biological macromolecules (Olafson and Sim, 1979; Olafson et al., 1979). It was immediately counted for 109 Cd and 65 Zn radioactivity and then digested in Soluene-350 (Packard, U.S.A.) at 60 ◦ C until clear. Following cocktail (Wallac OptiPhase HisSafe3) addition, the 35 S radioactivity associated with MTLP was ␤-counted. The concentrations of 109 Cd, 65 Zn and 35 S were calculated based on the whole wet tissue weight. The assimilation efficiencies (AEs) of Cd and Zn from T. weissflogii were quantified by a pulse-chase feeding technique. T. weissflogii was cultured in f /2 medium minus EDTA, Zn and Cu, and was labeled with 0.97 ␮Ci 109 Cd L−1 and 1.77 ␮Ci 65 Zn L−1 . Following 4 days of growth, the filtered algae were rinsed in nonradiolabeled seawater at least 3 times to minimize desorption of loosely cell-bound metals during the subsequent feeding period. Each scallop was kept in a polypropylene beaker containing 0.22 ␮m-filtered seawater. The dual-labeled algae were added into each beaker, and the animal was allowed to feed for 30 min. After that pulse feeding the scallop was immediately rinsed with clean seawater and measured for radioactivity. It was then transferred to clean seawater for 48 h of depuration, during which the scallops were periodically measured for radioactivity and the seawater and clean food were refreshed. The AEs of the metals were calculated as the percentage of the ingested 109 Cd and 65 Zn retained in the animals when the depuration curve began to level off. The elimination of Cd and Zn and MTLP degradation were quantified in radiolabeled scallops. Previously, it was found that the exposure pathway (e.g., dietary and waterborne) and radiolabeling duration (e.g., 12-h vs. 6-d) had little effect on metal efflux rate constant (except Ag) in the scallops (Pan and Wang, 2008a,b, 2009b) and the mussel Mytilus edulis (Wang et al., 1996). The animals were radiolabeled in 0.22 ␮m-filtered seawater spiked with 109 Cd (0.16 and 1.07 ␮Ci L−1 for the 8 and 40-d Cd exposed scallops, respectively), 65 Zn (0.61 and 1.20 ␮Ci L−1 for the 8 and 40-d Cd exposed scallops, respectively) and 35 S (8.72 and 2.50 ␮Ci L−1 for the 8 and 40-d Cd exposed scallops, respectively) for 1 day and then depurated in a clean environment for 16 days. The radioactivity in their bodies was measured at frequent intervals, and the seawater and their food were refreshed regularly. The scallops were sampled at days 0, 4, 8 and 16 to investigate MTLP degradation and the elimination of Cd and Zn associated with MTLPs. The natural logarithm of the percentage of 109 Cd or 65 Zn retained was regressed against the depuration time (d) and the absolute value of the slope was taken as the efflux rate constant (ke ) of the metal.

2.5. Radioactivity measurement and statistical analysis 109 Cd, 65 Zn, 110m Ag and 35 S radioactivity were measured with a Wallac ␥-counter (Perkin Elmer) at 88, 1115 and 658 keV and with a Wallac ␤-counter (Perkin Elmer). Counting times were adjusted to yield a propagated counting error of less than 5%. The spillover of radioisotopes was corrected for, and all counts were corrected for radioactive decay and any interference between ␥ and ␤ emissions. SPSS version 13.0 for Windows was used for all data analyses. To stabilize the variance and fulfill the normality assumption involved in analysis of variance, base 10 logarithmic or arcsine transformations were applied. Statistical significance was tested by an analysis of variance followed by the Student–Newman–Keul test. Difference were considered significant at values of p < 0.05.

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3. Results 3.1. Cd body concentration The Cd concentrations in the scallops’ soft tissues are reported in Table 1. After either 8 or 40 days of consuming Cd-spiked algae, the Cd concentration in the scallops was significantly elevated in a dose dependent manner. For example, after 8 days of Cd exposure the body Cd level increased by 7.4 times in the scallops feeding on 98 ␮g Cd g−1 algae and by 12.5 times with the 196 ␮g Cd g−1 algae. When exposed to algae at 58 ␮g Cd g−1 for 40 days the Cd concentration increased by 2.7 times, and by 4.2 times with 115 ␮g Cd g−1 algae. The background Cd concentrations in the two groups of control scallops (2.3 and 3.3 ␮g Cd g−1 wet weight) were higher than the EU import limit but below the USA limit. 3.2. MTLP induction and turnover The standing stock MTLP concentrations in the scallops are also shown in Table 1. After 8 days of dietary Cd exposure, the MTLP concentrations in the scallops doubled compared to the controls, but medium and high exposure led to comparable levels. After 40 days of Cd exposure the MTLP concentration remained constant at the control level (33.5 ␮g MTLP g−1 wet tissue), which was markedly higher than that in the other controls (3.0 ␮g MTLP g−1 wet tissue). ␤-Emitting cysteine, L-[35 S]-, was used to trace the synthesis and degradation of MTLP, as the proteins are rich in sulfur-bearing cysteine (Patierno et al., 1983; Krezoski et al., 1988; Ng et al., 2007). MTLP turnover in the scallops is shown in Fig. 1. The radioactivity of 35 S associated with MTLPs increased rapidly during the first 4 h regardless of the treatment, suggesting active de novo synthesis of the proteins. After that, the values leveled off in the control scallops but still progressively increased in the Cd-treated scallops over 8 days. Similar MTLP synthesis rate was found among the animals exposed to Cd for 40 days. Loss of L-[35 S]- was satisfactorily described by an exponential decay curve, and MTLP degradation was little affected by any dietary Cd treatment. The concentration of 35 S associated with MTLPs halved after the first 8 days of depuration. 3.3. Dynamics of MTLP-bound Cd and Zn ␥-Emitting 109 Cd and 65 Zn were used to trace the dynamics of Cd and Zn associated with MTLP, and the results are presented in Fig. 2. Over the 12 h uptake period, Cd and Zn radioactivity increased linearly with time. The rates of sequestration of new Cd and Zn by MTLP were comparable in treatments of either 8 or 40 days’ exposure. Over the 16 day metal depuration period the concentration of MTLP-bound 109 Cd remained constant, whereas 65 Zn was progressively lost from all of the scallops. About 25–74% of the MTLP-bound 65 Zn was depurated by the 16th day, but there was no significant difference in the elimination rate of MTLP-bound Zn among the treatments. 3.4. Biokinetics of Cd and Zn The Cd and Zn biokinetic parameters (dissolved uptake rate, assimilation efficiency and elimination rate) are reported in Table 1 and Fig. 3. Upon 8 days of exposure to Cd-enriched algae, the Cd AEs decreased from 90% to 75–78% while the Zn assimilation did not change. The scallops pre-exposed to algae containing 98 ␮g Cd g−1 showed a significantly higher dissolved Zn uptake rate than the controls and those pre-exposed to algae containing 196 ␮g Cd g−1 . Growing on Cd-enriched food had little effect on the rates of Cd and Zn elimination by the scallops, but Zn was depurated faster than Cd. Since no MTLP induction was found in the scallops exposed for 40

days, the AE and elimination of the metals were not quantified. Dissolved uptake of both Cd and Zn was little affected by the 40-day dietary Cd treatments (Table 1). 4. Discussion The main objective of this study was to investigate whether or not MTLPs regulate the uptake and elimination of Cd and Zn in marine bivalves following dietary Cd exposure, and if so how. In our previous study of this bivalve we found that MTLPs showed different roles in Cd uptake and elimination following waterborne Cd exposure (Liu and Wang, 2011). Specifically, increased MTLP induction did not affect Cd uptake from either the dissolved or the dietary phase, but instead resulted in significantly slower Cd elimination (Liu and Wang, 2011). This study focused on dietary Cd exposure, which is the dominant Cd accumulation pathway in the scallops (Pan and Wang, 2008a). MTLP induction may respond differently to Cd exposure through water or food (Amiard et al., 2006). The present results further confirm that MTLP induction is decoupled from Cd uptake and Cd recycling within MTLPs. Interestingly, the induction and turnover of MTLPs after dietary Cd exposure were totally different from those induced by waterborne Cd exposure, and the role of MTLPs in Zn biokinetics differed from that in Cd biokinetics. 4.1. Influences of dietary Cd exposure on MTLP induction and turnover One physiological role of MTLPs is to sequester excess intracellular Cd in response to metal stress (Nordberg, 1998; Klaassen et al., 1999; Nordberg, 2009). However, this was not the case when C. nobilis was exposed to dietary Cd. Following 8 days of exposure to 98 ␮g Cd g−1 food the scallops’ stock of MTLPs had only doubled and the induced MTLPs sequestered less than 5% of the accumulated Cd. With an increase in the dietary Cd concentration to 196 ␮g Cd g−1 there was no further increase in MTLP induction but a strong rise in the scallops’ body Cd concentrations. These results suggest that the bivalve could not induce enough Cd-binding proteins in the face of extreme dietary Cd challenges. This is consistent with the results obtained after the chronic dietary Cd exposure. Specifically, no induction of MTLP was found after 40 days of dietary Cd exposure (at 58 ␮g Cd g−1 or 115 ␮g Cd g−1 ) despite much greater Cd bioaccumulation. In line with these findings, a minor role for MTLPs in Cd sequestration has been reported in studies of several other bivalves such as Anondonta cygnea, Ostrea edulis, Scrobicularia plana and Unio elongatulus (Langston et al., 1998). In contrast, MTLP concentrations increased 4.5–20-fold after exposure to waterborne Cd, and 30% of the accumulated Cd was sequestered by the proteins (Liu and Wang, 2011). So the mode of exposure plays an important role in MTLP induction, even though the Cd body concentrations induced by dietary and waterborne Cd exposure are comparable. Studies of the mussel M. edulis have similarly shown that MTLP induction by dissolved Cd was more significant than that by dietary Cd exposure (Géret et al., 2000; Amiard et al., 2006). MTLP turnover reflects the net product of MTLP synthesis and subsequent degradation (Couillard et al., 1995; Wang and Rainbow, 2010). Upon metal stress, increased MTLP turnover would probably improve the animal’s ability to detoxify accumulated metals. Irrespective of dietary treatment, all of the scallops in this study showed active MTLP synthesis and relatively slow degradation of the newly synthesized proteins. Exposure to 98 ␮g Cd g−1 in the diet raised the MTLP synthesis rate, but an increase in dietary Cd concentration to 196 ␮g Cd g−1 did not raise it further. Intracellular Cd bioaccumulation thus appears to stimulate MTLP synthesis, but the rate is independent of the body Cd concentration. The limit on

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A 8d

D0.5 D98 D196

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35S in MTLPs (pmol g-1)

365

.1

.1

0.0

0.0

.15

.15

D0.5 D58 D115

.10

8d

.2

40 d

40 d .10

.05

.05

0.00

0.00 0

4

8

12

Uptake time (h)

16

0

4

8

12

16

20

Depuration time (d)

Fig. 1. Biosynthesis (A) and degradation (B) of metallothionein-like proteins in Chlamys nobilis pre-exposed to dietary Cd for 8 or 40 days. D: dietary exposure; numbers following ‘D’ indicate the Cd concentrations in the food. Data are mean ± standard deviation (n = 4 or 5).

MTLP synthesis under high metal stress might be partly explained by a general toxic effect of the Cd on synthesis of MTLPs (Barka et al., 2001; Amiard et al., 2006). Dietary exposure for 40 days did not improve MTLP synthesis, and new proteins were apparently synthesized only within the first 4 h. In contrast, 15 days of waterborne Cd exposure resulted in a linear increase in MTLP synthesis (Liu and Wang, 2011). The relatively slow protein synthesis observed further demonstrates the minor detoxifying role of MTLPs in scallops chronically exposed to dietary Cd. On the other hand, dietary Cd exposure did not affect the MTLP degradation rate, and half of the newly produced protein had been broken down after 8 days. The minimal effect of Cd exposure on MTLP degradation could be because degradation was primarily regulated by intracellular Zn content (Chen and Failla, 1989) rather than by Cd levels. 4.2. MTLP and Zn/Cd biokinetics The significance of MTLP in metal biokinetics has been demonstrated in several aquatic species (Ng et al., 2007; Serafim and Bebianno, 2007; Wang and Rainbow, 2010), but few studies have attempted to examine the relationships between MTLP turnover and metal biokinetics. In agreement with previous findings (Liu and Wang, 2011), Cd uptake was little affected by induced MTLP and de novo synthesis of the proteins. After 8 days of dietary Cd exposure both body MTLP concentration and newly synthesized protein were significantly enhanced, but the rate of Cd uptake from the dissolved phase did not change. The same situation has been reported for Cd-contaminated Perna viridis mussels (Blackmore and Wang, 2002; Shi and Wang, 2005). It seems reasonable if MTLP stoichiometry (the number of available binding sites) is taken into account. In general, one protein molecule can bind at most six Cd atoms in invertebrates (Hunt et al., 1984). In this study the ratio was lower than 1:6, so the induced MTLP had little effect on sequestration of newly accumulated Cd. Furthermore, the rate of Cd incorporation into MTLPs was similar in all of the scallops, even though the animals in the 8-d group were more active in de novo synthesis of MTLPs. The decoupling of dissolved Cd uptake from MTLP biosynthesis was further confirmed by the results with the 40-d group. MTLPs are considered to be actively involved in the assimilation of dietary Cd on the basis of significant correlations between MTLP

induction and the AEs of metals observed with several marine animals (Blackmore and Wang, 2002; Shi and Wang, 2004; Long and Wang, 2005). In the clam R. philippinarum and the green mussel P. viridis, for example, higher MTLP levels tend to be associated with higher Cd AEs from the dietary phase (Blackmore and Wang, 2002; Shi and Wang, 2004; 2005). But a negative relationship between MTLP concentration and Cd AE was found in the scallops (Table 1). MTLPs actually made little contribution to the reduced Cd assimilation, because the amount of Cd sequestered by induced MTLPs was negligible. Regardless of the role of MTLPs in metal assimilation, reduced assimilation of dietary Cd can certainly make the species more tolerant of metal pollution. Decho and Luoma (1996) have similarly reported that the bivalve Potamocorbula amurensis is capable of modifying its digestive process to reduce its interaction with high chromium concentrations. The influence of MTLPs on Zn uptake has been much less studied in aquatic invertebrates, although it is well documented that Zn is a constitutive metal of the protein (Klaassen et al., 1999; Amiard et al., 2006). We hypothesized that enhanced MTLP induction can simultaneously stimulate Zn uptake, since the high affinity of free sulfhydryl groups for Zn could efficiently reduce intracellular concentrations of the free metal. In fact the dissolved Zn uptake rate increased by 22.4% in the scallops exposed to a 98 ␮g Cd g−1 diet for 8 days over the rate observed in the controls (Table 1). The sequestration rate of newly incoming Zn by MTLPs, however, was not significantly enhanced (Fig. 2). So the slightly increased Zn uptake rate was not due to the up-regulation of MTLP expression. Furthermore, the Zn uptake rate of animals exposed to 196 ␮g Cd g−1 algae was similar to that of the controls (Table 1). One explanation for the discrepancy could be that most free sulfhydryl groups had been bound by the accumulated Cd, so any induced MTLP had little effect on the sequestration of newly accumulated Zn. On the other hand, the enhanced MTLP induction had little effect on Zn’s AE in the species. Degradation of MTLPs is also important in regulating intracellular transport and depuration of metals in marine bivalves (Serafim and Bebianno, 2007; Pan and Wang, 2008b, 2009a; Liu and Wang, 2011). MTLP-metal complexes are degraded in both lysosomal and non-lysosomal cell compartments where metals are released from the proteins (Chen and Failla, 1989). Large differences in the rate

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A

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Depuration time (d)

Fig. 2. Dynamics of Cd and Zn associated with metallothionein-like proteins over the period of dissolved uptake (A) and elimination (B) of the metals by Chlamys nobilis exposed to dietary Cd for 8 or 40 days. D: dietary exposure; numbers following ‘D’ indicate the Cd concentrations in the food. Data are mean ± standard deviation (n = 4 or 5).

of elimination of body metals may partly result from differences in the fate and release rate of MTLP-bound metals. The concentration of MTLP-bound 109 Cd remained constant while MTLP-bound 65 Zn was progressively depurated from the protein pool (Fig. 2), although MTLPs themselves have a relatively short half-life of about 8 days (Fig. 1). Most likely any Cd released from protein was immediately sequestered by newly biosynthesized MTLPs but most of the Zn was slowly depurated. This could be the reason why Cd had a longer half-life (approximately 77–116 days) in these scallops than Zn did (17–23 days). Interestingly, a previous study in our laboratory found that enhanced MTLP induction could even result in Cd translocation from other intracellular compartments to the MTLP pool, finally leading to a slower elimination rate of the metal in those with higher MTLP levels (Liu and Wang, 2011). 4.3. MTLPs as biomarkers The concentration of MTLPs in the scallop tissue was not a good biomarker for Cd exposure because of the limited MTLP induction after dietary Cd exposure. MTLP concentrations in the bodies of scallops in the two control groups (8 days and 40 days of expo-

sure) showed large differences (Table 1), and these levels (3.0 and 33.5 ␮g g−1 wet wt.) were also much higher than those measured in a previous study (0.4 ␮g g−1 wet wt., Liu and Wang, 2011). This can be mainly attributed to the different animal sizes used in the different experiments. Indeed, a significant negative relationship between MTLP concentration and body wet weight was evident in the uncontaminated scallops (r2 = 0.89, p < 0.0001, n = 17, Fig. 4). The allometric effect of body size on MTLP concentration has been also observed in the periwinkles Littorina littorea (Leung and Furness, 1999) and rats (Klaassen et al., 1999). The mechanism underlying the consistently higher levels of MTLPs in smaller individuals remains unknown, but when evaluating environmental pollution using MTLP level as a biomarker, size clearly needs to be considered. At the same time, these experiments were performed under laboratory conditions over 8 or 40 days. It may be that factors such as temperature, food and salinity may influence the induction of MTLPs in these animals (Amiard et al., 2006). Overall, any factor rather than metal contamination affecting MTLP levels would limit their utility as a biomarker of metal exposure. Increased MTLP turnover may reflect environmental Cd contamination with greater sensitivity than MTLP stocks (Couillard et al.,

F. Liu, W.-X. Wang / Aquatic Toxicology 105 (2011) 361–368

A

C

B 100

Cd retained (%)

D0.5 D98 D196

60

75

0

0

10

.50

100

100

0

4

8

12

Uptake time (h)

109

75 50 25 0

16

Zn retained (%)

65

0.00

25

65

Zn retained (%)

.25

Tissue

65

109

30

-1

Tissue

50

100

Zn (μg g )

109

Cd retained (%)

-1

Cd (ng g )

90

367

0

10

20

30

40

50

Depuration (h)

60

10

0

4

8

12

16

Depuration time (d)

Fig. 3. Dissolved uptake (A), assimilation efficiency (B) and elimination (C) of Cd and Zn by Chlamys nobilis exposed to dietary Cd for 8 days. D: dietary exposure; numbers following ‘D’ indicate the Cd concentrations in the food. Data are mean ± standard deviation (n = 4–10).

References

60

-1

MTLP (μg g )

2

R = 0.89 P < 0.0001

40

20

0 0.0

0.5

1.0

1.5

2.0

Body wet weight (g) Fig. 4. Total MTLP concentration and the body wet weight of uncontaminated Chlamys nobilis scallops. Each data point represents one individual scallop, and the data include 8 clean individuals from our previous study (Liu and Wang, 2011).

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