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Metal (Ag, Cd, Cu, Ni, Tl, Zn) binding to cytosolic biomolecules in field-collected larvae of the insect Chaoborus Maikel Rosabal, Sandra Mounicou, Landis Hare, and Peter G.C. Campbell Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b05961 • Publication Date (Web): 17 Feb 2016 Downloaded from http://pubs.acs.org on February 27, 2016

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Environmental Science & Technology

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Metal (Ag, Cd, Cu, Ni, Tl, Zn) binding to cytosolic biomolecules in field-

2

collected larvae of the insect Chaoborus

3 4 5 6

Maikel Rosabala, Sandra Mounicoub*, Landis Harea and Peter G.C. Campbella

7 8

a

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(INRS-ETE), 490 de la Couronne, Quebec City, QC G1K 9A9, Canada

Institut National de la Recherche Scientifique – Centre Eau Terre Environnement

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b

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UMR 5254, Hélioparc, 2. Av. Pr. Angot, Pau, 64053 France

CNRS/UPPA, Laboratoire de Chimie Analytique Bio-Inorganique et Environnement

13 14 15 16

*

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Pau, 64053 France, phone: ++335 59 40 77 64, email: [email protected].

Corresponding author: CNRS/UPPA, LCABIE UMR 5254, Hélioparc, 2. Av. Pr. Angot,

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Abstract

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We characterized the biomolecules involved in handling cytosolic metals in larvae of

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the phantom midge (Chaoborus) collected from five mining-impacted lakes by

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determining the distribution of Ag, Cd, Cu, Ni, Tl and Zn among pools of various

25

molecular weights (HMW: high molecular weight, > 670 kDa – 40 kDa; MMW: medium

26

molecular weight, 40 kDa – 670 kDa” or “< 1.3 kDa” for SEC200 and as “> 10 kDa” for

114

SECpep. Molecular masses for biomolecules eluting outside the column calibration ranges

115

cannot be calculated from the calibration curve, which is not linear outside these limits,

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and cannot be compared between the two chromatographic columns. As no biomolecules

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elute before the void volume of the column, chromatograms are presented after 10

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minutes of acquisition time in order to improve the data presentation.

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The chromatographic effluent was then introduced into the MWD for evaluation of

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protein absorbance at 280 nm and 254 nm before subsequent delivery into the ICP-MS

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for 60Ni, 64Zn, 65Cu, 107Ag, 114Cd and 205Tl measurements. Larvae from three lakes

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(reference lake Opasatica; contaminated lakes Swan and Lohi) were used to screen for

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metal-binding biomolecules in the whole cytosol, whereas larvae from all lakes were used 6 ACS Paragon Plus Environment

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for analysis of the HSP fraction. To identify the proportion of cytosolic metal in each

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SEC200 MW pool (HMW, MMW and LMW), the ratio of the peak area of each MW pool

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to the sum of the total chromatogram area was calculated. Similarly, the variation of the

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MW distribution of metals in the HSP fraction, as a function of the total metal in this

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fraction, was determined by plotting the peak area of each fraction (normalized by total

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larval dry weight) against the total metal concentrations in the HSP fraction.

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2.4

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Metal quantification and controls The homogenate and all fractions (cytosol, pellet, HSP and HDP) were digested (70%

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nitric acid, 80°C, 2 h) for total metal content determination by external calibration using

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standard concentrations ranging from 0.1 to 10 µg L-1 (Ni, Ag, Cd and Tl) and from 1 to

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100 µg L-1 (Cu and Zn) in 2% nitric acid. .Metal mass balances (mean value ± SD, n =

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15) were then calculated (details in SI) and acceptable values were obtained for Ni (111%

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± 36%), Zn (107% ± 35%), Cu (107% ± 31%), Ag (92% ± 24%), Cd (111% ± 20%) and

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Tl (111% ± 23%). Acceptable HPLC system recoveries (including injector, tubing and

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column – details in SI) using the SEC-200 column were obtained for Ni (106% ± 22%, n

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= 6), Zn (137% ± 7%, n = 5), Cu (126% ± 9%, n = 5), Ag (101% ± 38%, n =5), Cd

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(110% ± 3%, n = 6) and Tl (97% ± 9%, n = 6).

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2.5

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Statistical analysis All numerical data are represented by means ± standard deviations (SD). Differences

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in fraction area among the cytosolic MW pools (% values, after arcsine transformation)

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were assessed by one-way ANOVA using a Tukey honest significant differences (HSD)

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test. For the HSP fraction, relationships between total metal concentrations and the

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SECpep fraction area of each metal (normalized by total larval weight) were initially 7 ACS Paragon Plus Environment


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examined in bivariate scatterplots and tested by simple correlation analysis (Pearson r)

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after verifying assumptions of normality (Shapiro-Wilk test). When bivariate plots

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indicated a possible relationship, linear and exponential regression models were tested

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when the necessary assumptions were satisfied. Correlations reported between fraction

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areas of metals were obtained by applying non-parametric Spearman rank correlations.

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Statistical analyses were performed using STATISTICA version 6.1 software (StatSoft,

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Tulsa, Oklahoma, USA) and P-levels of 0.05 were used as the threshold for statistical

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significance.

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3

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3.1

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Results and Discussion Metal bioaccumulation gradients Metal concentrations in the cytosol and the HSP fraction of Chaoborus larvae varied

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greatly among lakes (Table 1). Higher cytosolic metal concentrations were found in

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larvae collected from Lakes Lohi (Ag, Cd, Cu, Ni and Zn) and Swan (Tl) compared to

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those from Lake Opasatica, where the lowest metal concentrations were measured

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(excluding Zn). With regard to the HSP fraction, Chaoborus from Lakes Lohi (Ni, Zn,

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Cu, Ag), Tilton (Cd) and Swan (Tl) had the highest metal concentrations, whereas those

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from Lake Opasatica again had the lowest metal levels. Ratios of maximum to minimum

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metal concentrations ([M]max / [M]min) for both fractions covered a broad range, with

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higher ratios for the non-essential metals (e.g., Ni, Cd) than for the essential metals (Cu,

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Zn). As a consequence of the Ni-rich emissions from the smelters located in Sudbury15,

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but not Rouyn-Noranda, the highest [M]max / [M]min ratios were observed for Ni (cytosol:

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33; HSP: 24). Large differences between the least and the most contaminated fractions

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were also found for Cd (cytosol: 18; HSP: 21) and Tl (cytosol: 5.0; HSP: 4.7), whereas 8 ACS Paragon Plus Environment

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the bioaccumulated metal gradient was lower for Cu (cytosol: 3.0, HSP: 2.5) and Zn

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(cytosol: 2.4, HSP: 3.3). These results are in agreement with previous studies on

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Chaoborus collected in the field along metal exposure gradients, where larval Cu and Zn

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concentrations varied less than those of the non-essential metals (e.g., Cd; Ni).14 At the

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subcellular level, metals measured in the HSP fraction represented more than 50% (n =

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12-15) of the total larval burden for Ag (50% ± 23%), Cd (80% ± 17%), Cu (75% ±

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32%), Ni (65% ± 38%) and Tl (79% ± 19%). These proportions are similar to those

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previously reported for field-collected Chaoborus for which the majority of the

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bioaccumulated Cu, Cd and Ni was found in the HSP fraction.14

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3.2

Metal binding to cytosolic ligands

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The cytosolic fractions of Chaoborus larvae collected from Lakes Opasatica,

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Swan and Lohi were analyzed by SEC200-ICP-MS to perform a broad screening of the

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cytosolic metal-containing biomolecules (Figs. 1, 2, S2). Metal-binding biomolecule

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fractions were operationally defined as HMW (high molecular weight; > 670 kDa – 40

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kDa; elution time: 10-22.5 min), MMW (medium molecular weight; 40 kDa – < 1.3 kDa;

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elution time: 22.5-32 min) and LMW (low molecular weight; ˂ 1.3 kDa; elution time: ˃

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32 min). The UV detection data at 280 nm (data at 254 nm are not shown as they are

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similar) show that biomolecules elute over the entire molecular mass range of the

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column, with some overlap with the metal elution peaks.

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3.2.1

Copper and zinc

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For the two essential metals studied, much higher proportions were found in the

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MMW pool (average for all lakes of 79% ± 1.4% for Zn and 99% ± 0.1% for Cu, n = 9)

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than in the HMW pool (Fig. 2). Similarly, in liver of gibel carp (Carassius auratus

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gibelio) collected from metal-impacted habitats, the Cu and Zn concentrations in the

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MMW pool (defined as ~10 kDa) also increased as a function of the cytosolic metal

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concentrations.19 Copper and Zn binding to MMW ligands was also identified in

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zebrafish gills after uranium exposure, where Zn was also associated with high MW

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proteins.20 The appreciable Zn signals measured in the HMW and MMW fractions in

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Chaoborus could be associated with the role of this essential metal in the stability of zinc-

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finger structures21 reported in various dipteran proteins located in the cytosol, including

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E3 ubiquitin-protein ligase (130 kDa)22, ubiquitin protein ligase deltex (82 kDa)23 and

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translation inhibition factor 2 (35 kDa).24 With regards to Cu, its modest presence in the

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HMW pool likely reflects the association of Cu with physiologically important

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metalloproteins.25 Furthermore, both of these essential metals were almost certainly

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associated with MT or MTLP (5-4.0 kDa) since these proteins are known to be involved

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in the homeostasis of these elements.7

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3.2.2

Cadmium

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Virtually all of the cytosolic Cd was found in the MMW pool (average for all larval

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samples of 97 ± 1.2 %, n = 9), irrespective of the actual Cd concentration in the cytosol

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(Figs. 1B, C; 2). The association of Cd with MTLP bioligands in Chaoborus has been

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reported previously in a study where MTLP concentrations were determined with a Hg-

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saturation method.26 These authors obtained a strong correlation between the measured

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MTLP and total larval Cd concentrations, reflecting the induction of MTLP by Cd and

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the subsequent complexation (for detoxification purposes) of Cd by the induced peptide.

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High proportions of the total cytosolic Cd were also found in the MMW fraction in 10 ACS Paragon Plus Environment

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Anguilla anguilla27 (defined as 10 – 20 kDa) and in C. auratus gibelio19 (defined as ~10

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kDa) collected from metal-impacted habitats, and this accumulation increased with

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increasing cytosolic Cd concentrations. The predominant role of MTLP in sequestering

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Cd was also reported for indigenous floater mussels (Pyganodon grandis)28, where

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cytosolic Cd concentrations in the digestive gland were significantly related to the MTLP

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levels.

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3.2.3

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Silver

Higher proportions of total cytosolic Ag were found in the MMW pool than in the

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HMW pool (Fig. 2). At a low cytosolic Ag concentration (i.e., larvae from Lake

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Opasatica), Ag could only be detected in the MMW pool (Figs. 1B and 2). In cytosols

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from larvae originating from the two most contaminated lakes, Ag was bound to both

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HMW and MMW bioligands; the proportion of Ag in the MMW pool decreased from

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100% to about 79% in the cytosols of larvae collected from the two contaminated lakes

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(Fig. 2). High Ag concentrations in the MMW (10 – 20 kDa) pool compared to the HMW

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(˃20 kDa) pool were also found in the livers of Anguilla anguilla eels collected from

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sampling sites differing in their metal contamination levels.27 In eels, the percentage of

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Ag found in the MMW fraction, which represented up to 92% of the total cytosolic Ag,

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increased as a function of the Ag concentrations in the surrounding sediment and water.

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These observations are consistent with the preference of this “soft” metal for the

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sulfhydryl groups found in MT or MTLP.29 Other sulfhydryl-containing proteins in the

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HMW pool may also be targeted by this non-essential metal.

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3.2.4

Nickel

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The mean proportion of Ni binding to HMW ligands (Fig. 2) (including

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metalloenzymes) was approximately one-third of the total cytosolic Ni in Chaoborus

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collected from the two Ni-contaminated lakes (Lohi and Swan), which was significantly

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higher (P ˂ 0.05) than that for larvae from the reference lake (Opasatica). This result is

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consistent with observations reported for the amphipod crustacean Gammarus fossarum,

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where Ni in the “non-MTLP” pool (HMW (255 kDa – 18 kDa) plus LMW (˂ 1.8 kDa))

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increased significantly as a function of Ni exposure.10

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Most (69-78%) of the total cytosolic Ni was found in the MMW pool, and these

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proportions did not change (P ˃ 0.05) with increasing total cytosolic Ni (Fig. 2).

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Considering that Ni has a low affinity for sulfhydryl functional groups, its sequestration

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in the MMW pool is likely determined more by the occurrence of O- and P-containing

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ligands than by the presence of MT and other sulfhydryl-rich ligands.7 Based on the

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ability of Ni to replace essential metals in physiologically important enzymes30, the Ni

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peaks eluting from 23 min (16.5 kDa) to 30 min (1.3 kDa) may reflect binding of this

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element to medium molecular weight proteins.

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3.2.5

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Thallium

In contrast to the dominance of the HMW and MMW fractions for the other metals,

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Tl was only found in the LMW fraction (Figs. 1 and 2) where free amino acids and small

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peptides are found. Considering the potential interaction of Tl(I) with sulfhydryl-

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containing groups7, the partitioning of this metal could be ascribed to its association with

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free amino acids containing such chemical groups (e.g., cysteine).

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3.3

Metal binding to metallothionein-like peptides and other thermostable ligands

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To further investigate metal-binding to cytosolic biomolecules, we isolated the HSP

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fraction of larvae from five lakes by heat treatment of the cytosol. These HSP fractions

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were subjected to further separation using SECpep-ICP MS (Figs. 3,4 and S3). The metals

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studied were associated with various subcellular fractions operationally defined as SECpep

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fraction 1 (> 10 kDa – 6.2 kDa; elution time: 11-16 min), SECpep fraction 2 (6.2 kDa –

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1.7 kDa; elution time: 16-22 min) and SECpep fraction 3 (< 1.7 kDa; elution time: > 22

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min).

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3.3.1

Essential metals

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Within the HSP samples, more than 80% of the total Cu was found in MW SECpep

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fraction 2, with a peak apex at 17.4 min; both fractions 1 and 2 responded significantly

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along the total Cu bioaccumulation gradient in the HSP fraction (Fig. 4). In contrast, we

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observed that Zn measured in both SECpep fractions did not respond even though the total

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Zn concentration in the HSP fraction did increase (Fig. 4), suggesting an important loss

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of this metal during the chromatographic separations. Indeed, since we did not observe

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such a loss of Zn in the chromatographic separation of the cytosol, we speculate that the

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heat denaturation step may have led to the liberation of some inorganic Zn in the HSP

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fraction (e.g. Zn2+) which would be trapped on the stationary phase of SECpep column.

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Because of the apparent Zn loss in the HSP fraction, the data cannot be interpreted for

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SECpep fractions 1 and 2.

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3.3.2

Non-essential metals

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Cadmium in the HSP fraction from Lake Opasatica showed two chromatographic

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peaks at 17.4 min (4.7 kDa) and 18.5 min (3.2 kDa) in SECpep fraction 2, peaks denoted

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as Cd17.4min and Cd18.5min, respectively (Fig. 3B). Similar chromatographic profiles

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showing co-elution with Cd at these retention times were obtained for Ag, Cu and Zn in

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the HSP fractions of larvae originating from the reference lake (Fig. 3B). As the total Cd

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concentration in the HSP fraction increased, the Cd18.5min peak tended to disappear,

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whereas the Cd17.4min peak signal significantly increased (Figs. 3C and 4) obscuring the

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Cd18.5min peak. As was observed for the HSP samples collected from the reference lake,

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the chromatograms for HSP samples from the contaminated lakes also showed co-elution

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of Ag, Cu and Zn under the Cd17.4min peak (Fig. 3C).

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In addition to the peaks occurring in SECpep fraction 2, Cd was also detected in a

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biomolecule of higher molecular weight in the HSP samples from all lakes but the peak

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intensity levels were extremely low in the HSP samples from Lake Opasatica, the

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reference lake (Fig. S4). This Cd peak was detected at an elution time of 15.5 min,

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corresponding to a MW of 6.5 kDa (denoted as Cd15.5min in Fig. 3C). With increasing total

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Cd concentrations in the HSP fraction, the response of the biomolecules in SECpep

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fraction 2 (slope: 78 ± 4; P = 0.0005, n = 14) in sequestering this element was

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significantly stronger (P < 0.05) than that of SECpep fraction 1 (slope: 54 ± 6; P = 0.003,

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n = 14); as a consequence, Cd concentrations detected in SECpep fraction 2 were

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consistently higher than those in fraction 1 for all the larval HSP samples (Fig. 4).

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Our results suggest that at low Cd concentrations in the HSP fraction, this element is

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sequestered by two constitutive MT isoforms with molecular mass (estimated using data 14 ACS Paragon Plus Environment

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obtained from the calibration column) of ~4.7 kDa and 3.2 kDa, which are very similar to

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the MW reported for the MtnA and MtnB isoforms of the fruit fly Drosophila

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melanogaster.31, 32 When more Cd accumulates in the HSP fraction, the concentration of

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the putative 4.7 kDa MtnB isoform increases but the 3.5 kDa MtnA isoform does not

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respond in a similar manner. This observation agrees with previous studies33, 34 in which

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the existence of distinct MT isoforms playing different roles in metal detoxification was

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reported for snails. In addition to the putative 4.7 kDa MT isoform, biomolecules of

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higher mass (6.5 kDa, possibly MT dimers) also appear to be involved in Cd

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sequestration as the total Cd concentration increases in the HSP fraction. This metal-

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handling strategy under high Cd exposure is consistent with an earlier field study on

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chronically metal-exposed Chaoborus, where we demonstrated that the Cd detoxification

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response became more effective at higher internalized Cd concentrations.14

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Most of the Ag accumulated in the HSP fraction was found in SECpep fraction 2 (86%

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± 16% for all lakes, n = 12), which was found to increase as the total Ag concentration

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increased (Fig. 4). In contrast, Ag in SECpep fraction 1 did not increase as a function of

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the Ag accumulated in the HSP fraction (Fig. 4).

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The Ni profile of metal-containing biomolecules showed that the partitioning of this

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element occurred in SECpep fraction 1 (> 10 kDa – 6.0 kDa) and in SECpep fraction 3 (
670 kDa – 40 kDa; elution time: 10-22.5 min), MMW (medium molecular weight; 40 kDa – < 1.3 kDa; elution time: 22.5-32 min) and LMW (low molecular weight; < 1.3 kDa; elution time: ˃ 32 min). Figure 2. Peak area percentages (mean ± S.D.; n = 3) of 60Ni, 64Zn, 65Cu, 107Ag and 114

Cd in two cytosolic molecular weight pools (HMW, MMW) of Chaoborus

collected from Lakes Opasatica, Swan and Lohi. The cytosols were fractionated by SEC200-ICP-MS. Bars with the same letters indicate non-significant differences, whereas different letters indicate that the differences are significant (ANOVA followed by Tukey test, P < 0.05). Figure 3. SECpep chromatograms of metal-containing ligands in the HSP fraction of Chaoborus larvae, with UV detection at 280 nm for larvae from Lake Opasatica (black line, reference lake) and Lake Tilton (orange line, highly contaminated lake) (A), and with ICP-MS detection for larvae from Lake Opasatica (B) and from Lake Tilton (C). The three operationally-defined MW fractions comprise: fraction 1 (> 10 kDa – 6.2 kDa; elution time: 11-16 min); fraction 2 (6.2 kDa – 1.7 kDa; elution time: 16-22 min); fraction 3 (< 1.7 kDa; elution time: > 22 min). 27 ACS Paragon Plus Environment


Figure 4. Relationships between peak area (107 cps2) normalized for larval weight (g dw) (mean ± S.D.; n = 2-3) for 60Ni, 64Zn, 65Cu, 107Ag, 114Cd, 205Tl and the total metal concentration in the HSP fraction of Chaoborus larvae for the three MW SECpep fractions: fraction 1 (> 10 kDa – 6.2 kDa; elution time: 11-16 min); fraction 2 (6.2 kDa – 1.7 kDa; elution time: 16-22 min); fraction 3 (< 1.7 kDa; elution time: > 22 min).


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350

1.3 kDa

6.8 kDa

40 kDa 16 kDa

80 kDa

474 kDa


670 kDa

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A

Absorbance

300

HMW

250

MMW

LMW

200 150

Lake Lohi

100

Lake Opasatica

50 0 12

10

12

14

16

18

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22

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28

30

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36

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40

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46

48

50

52

Lake Opasatica

8 6

Intensity (x104 cps)

56

58

60

60Ni

B

10

54

64Zn

x 0.1

65Cu

x 0.1

107Ag

x7

114Cd

4

205Tl

2

x 20

0 120

10

12

14

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18

20

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50

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C

100

Lake Lohi

80 60 40

54

56

58

60Ni

x3

64Zn

x 0.5

65Cu

x 0.6

107Ag

x 24

114Cd

x 0.5

205Tl

60

x 20

20 0 10

12

14

16

18

20

22

24

26

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30

32

34

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38

40

Time (min)

Figure 1


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50

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54

56

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60


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120 Opasatica Swan Lohi c

Peak area %

100

Zn

b

Cu

Ag

b

b

Cd c

b

Ni 80

60

b a

a

40

a a

20

a 0 HMW

MMW

HMW

MMW

HMW

MMW

HMW

Cytosolic molecular weight fractions

Figure 2


MMW

HMW

MMW

1.3 kDa

10 kDa

A

0.12 kDa


6.8 kDa

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Absorbance (280 mn)

250

Fraction 1

Fraction 3

Fraction 2

200 Lake Tilton 150 Lake Opasatica 100 50 0 12

10

12

14

16

18

22

24

26

28

30

32

34

B

10

36

38

40

42

44

46

40

42

44

46

60Ni

Lake Opasatica

8

Cd17.4

6

Intensity (x104 cps)

20

64Zn

x 0.1

65Cu

x 0.1

107Ag

Cd18.5

x7

4

114Cd

2

205Tl

x 20

0 10 180

12

14

16

C

160

18

20

22

24

26

Cd17.4

28

30

32

34

36

38

Fraction 3-2

15

Lake Tilton

140

60Ni

10

120

64Zn

100

x 0.5

Fraction 1 5

65Cu

80

0

Cd15.5

60

Fraction 3-1

x 0.5 0

40

4

8

12 16 20 24 28 32 36 40 44 48

20 0 10

12

14

16

18

20

22

24

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28

30

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Time (min)

Figure 3


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40

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30

250

Ni

25

Area larval weight-1 (x107cps2g-1dw)

Fraction 1 Fraction 3-1 Fraction 3-2

350

Fraction 1 Fraction 2

Cu

300

20

250


Cd 200 150

200

15

100

150 10

100 5

50

0

0

50 0

0

1

2

3

4

5

6

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8

9

150 125

0

30

40

50

60

0

70

10

15

20

25

30

4

8

Ag


Zn

5


Fraction 3

Tl

6

3

4

2

2

1

100 75 50 25 0 100

150

200

250

300

350

400

0 0.05

0

0.10

0.15

0.20

0.25

[M] HSP fraction (nmol g-1 dw)

Figure 4


0.30

0.005

0.010

0.015

0.020

0.025

35


Graphical abstract

SEC-ICP-MS Fraction 1 Fraction 2

Centrifugation Heat treatment Centrifugation

Fraction 3 Ni

Homogenization

Intensity

Page 33 of 33

Cd Cu Ag Tl

Time

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