Mercury tissue residue approach in Chironomus riparius: Involvement of toxicokinetics and comparison of subcellular fractionation methods

doi:10.1016/j.aquatox.2015.11.027

Aquatic Toxicology

Volume 171, February 2016, Pages 1-8

https://doi.org/10.1016/j.aquatox.2015.11.027 Get rights and content

Highlights

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About 90% of the accumulated Hg in Chironomus riparius larvae was internalized in the cytosolic compartment.
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Metallothionein-like proteins (MTLP) sequestered about a third of the Hg flux entering the cytosol.
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The sensitive fraction (heat-sensitive proteins, HSP) progressively saturated leading to Hg excretion and physiological impairments.
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For Hg subcellular fractionation, the size exclusion chromatography separation (SECS) method is recommended rather that the heat-treatment and centrifugation (HT&C) method.

Abstract

Along with the growing body of evidence that total internal concentration is not a good indicator of toxicity, the Critical Body Residue (CBR) approach recently evolved into the Tissue Residue Approach (TRA) which considers the biologically active portion of metal that is available to contribute to the toxicity at sites of toxic action. For that purpose, we examined total mercury (Hg) bioaccumulation and subcellular fractionation kinetics in fourth stage larvae of the midge Chironomus riparius during a four-day laboratory exposure to Hg-spiked sediments and water. The debris (including exoskeleton, gut contents and cellular debris), granule and organelle fractions accounted only for about 10% of the Hg taken up, whereas Hg concentrations in the entire cytosolic fraction rapidly increased to approach steady-state. Within this fraction, Hg compartmentalization to metallothionein-like proteins (MTLP) and heat-sensitive proteins (HSP), consisting mostly of enzymes, was assessed in a comparative manner by two methodologies based on heat-treatment and centrifugation (HT&C method) or size exclusion chromatography separation (SECS method). The low Hg recoveries obtained with the HT&C method prevented accurate analysis of the cytosolic Hg fractionation by this approach. According to the SECS methodology, the Hg-bound MTLP fraction increased linearly over the exposure duration and sequestered a third of the Hg flux entering the cytosol. In contrast, the HSP fraction progressively saturated leading to Hg excretion and physiological impairments. This work highlights several methodological and biological aspects to improve our understanding of Hg toxicological bioavailability in aquatic invertebrates.

Introduction

The critical body residue (CBR) is the concentration of a chemical species bioaccumulated in an organism that corresponds to a defined onset of toxicity, regardless of how it is measured (e.g., mortality, growth inhibition…) (McCarty and Mackay, 1993). This approach has recently evolved into a more general concept, which considers tissue residues as the dose metric when characterizing dose-response relationships, evaluating mixture toxicity, developing guidelines to protect organisms, and conducting risk assessments (Sappington et al., 2011). The so-called tissue residue approach (TRA) relies on the toxicological principle that a toxic effect is not observed unless the chemical reaches the site of action, i.e., only a fraction of the contaminant taken up is metabolically available to interact with sites of toxic action (Rainbow, 2007). Concerning metals, both essential elements in excess of metabolic requirements and non-essential elements must be detoxified either via excretion or sequestration (bound to cytosolic proteins or low-molecular weight compounds and to ‘insoluble’ deposits) processes. The cytosolic proteins involved are generally metallothioneins (MT) or metallothionein-like proteins (MTLP), which can specifically bind certain trace metals (e.g., Ag, Cd, Cu, Hg, Zn) (Amiard et al., 2006). Insoluble deposits include relatively heterogeneous lysosomal residual bodies or commonly discrete metal-rich granules (MRG) (Mason and Jenkins, 1995, Marigómez et al., 2002). According to Wallace et al. (2003), MT and MRG can be grouped as biologically detoxified metal (BDM) and the cytosolic organelles and target molecules such as heat-sensitive proteins (HSP, mostly enzymes) can be grouped as metal-sensitive fraction (MSF), which are of particular interest when considering TRA for toxicity assessment. In 2011, the TRA Special Series of Integrated Environmental Assessment and Management (volume 7, issue 1, pages 1–154) demonstrated that the TRA remains intrinsically limited if it is targeted at determining steady-state, whole-organism concentrations but may further advance if it is focused on target site concentrations and if toxicokinetic (what the organism does with the toxicant) and toxicodynamic (what the toxicant does to the organism) characteristics are integrated (Adams et al., 2011).

The knowledge of the subcellular fate of metals is therefore a prerequisite to better understand physiological processes underlying their bioaccumulation and toxicity at various biological levels (Perceval et al., 2004, Campbell et al., 2005) as well as throughout the food web (Cheung and Wang, 2005, Seebaugh and Wallace, 2009). One of the most elaborate protocols of subcellular fractionation of metals was developed by Wallace and Lopez (1996) based on heat treatment and centrifugation (referred as HT&C method) allowing the partitioning of metals in the cytosolic fraction according to the heat-stable and heat-denaturation characteristics of MTLP and HSP, respectively. However, potential methodological artifacts may occur during this critical step, which are globally linked with the stability of the sulphur binding driving the association of metals with thiol compounds of the proteins (Bragigand and Berthet, 2003). Hence, alternative approaches using size exclusion chromatographic separation (referred to as SECS method) have been used to assess metal partitioning in the cytosolic fraction (Fraysse et al., 2006, Perceval et al., 2006). A comparative study of subcellular fractionation of cadmium (Cd), nickel (Ni) and lead (Pb) in Gammarus fossarum using both SECS and HT&C methods emphasized important differences in the cytosolic partitioning of Cd particularly but also of Ni and to a lesser extent of Pb between HSP and MTLP (Geffard et al., 2010).

Among studied metals known to have significant toxicological impact on biota, mercury (Hg) has been identified as a potent neurotoxicant for animals and humans, especially under its organometallic form (i.e., methylmercury) which strongly binds with sulphydryl groups in proteins and is therefore readily accumulated and retained in tissues (Clarkson, 1992). Up to date, only few data are available regarding its subcellular distribution in aquatic invertebrates (Seebaugh and Wallace, 2009, Xie et al., 2009, Pan and Wang, 2011). Moreover, these studies exclusively used the Wallace-based fractionation protocol and the issue regarding its efficiency to accurately define the cytosolic partitioning of Hg is still pending.

The main objectives of the present research were to study Hg bioaccumulation and fractionation in larvae of the midge Chironomus riparius. First, we compared the efficiency and accuracy of the two methodologies previously mentioned for subcellular fractionation of Hg both in terms of Hg balance recoveries and partitioning fluxes. Second, we used a modeling approach, using a toxicokinetic model, to assess environmental bioavailability (i.e., uptake and bioaccumulation processes) and toxicological bioavailability (i.e., the portion of assimilated chemical that reaches and interacts with the sites of toxic action). Finally, ecotoxicological implications are discussed.

Section snippets

Organisms

We used fourth instar larvae of C. riparius cultured in the laboratory prior to the experiments according to standard methods (details given in the Supplementary material I).

Sediment spiking

We spiked artificial silica sediment (TerraSand, JBL, Germany) previously sieved through a 400 μm mesh. The particle size distribution was 50% between 250 and 400 μm, and 50% below 250 μm (Coulter^® LS-100, Beckman Coulter, Fullerton, CA, USA). Seven kilograms of artificial sediments were first maintained with 6 L of Lake Geneva

Whole body and partitioning toxicokinetics of mercury

For C. riparius larvae exposed to control conditions (1.56 ± 0.06 ng g⁻¹ dry weight and 4.93 ± 0.13 ng L⁻¹ in sediments and overlying water, respectively), no increase of total Hg concentration was observed and internal concentrations never exceeded 0.05 μg g⁻¹ (fresh weight) in the entire body (homogenate (H) fraction, data not shown). Under the exposure conditions (1.35 ± 0.08 μg g⁻¹ dry weight and 6.99 ± 0.30 μg L⁻¹ in sediments and overlying water, respectively), total Hg concentrations in the homogenate (H)

Conclusions

The proposed toxicokinetic approach appears as an interesting tool, along with toxicodynamic considerations, for the development of more mechanistic approaches regarding sublethal toxicity and understanding species sensitivity. For this purpose, fractionation methods containing a heating step are not recommended, especially for trace metals subject to volatilization such as Hg or arsenic (As). Separation using size exclusion chromatography appears to be an accurate methodology and allows to

Acknowledgements

This study was supported by the Swiss National Science Foundation (grant no. 200020-117942). The authors sincerely thank Hervé Sartelet (Laboratoire Signalisation des Récepteurs Matriciels SiRMa, Université de Reims Champagne-Ardenne) for technical assistance in SECS and Benjamin Pauget (Department Chrono-Environment, University of Bourgogne Franche-Comté) for fruitful discussions about the manuscript.

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¹: ^{Present address: ISMAR-CNR, Arsenale—Tesa 104, Castello 2737/F, 30122 Venezia, Italy.}

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Mercury tissue residue approach in Chironomus riparius: Involvement of toxicokinetics and comparison of subcellular fractionation methods

Highlights

Abstract

Introduction

Section snippets

Organisms

Sediment spiking

Whole body and partitioning toxicokinetics of mercury

Conclusions

Acknowledgements

Aquat. Toxicol.

Comp. Biochem. Physiol. C

Anal. Chim. Acta

Comp. Biochem. Physiol. A

Aquat. Toxicol.

Mar. Chem.

Comp. Biochem. Physiol. C

Chemosphere

Environ. Int.

Environ. Pollut.

Aquat. Toxicol.

Aquat. Toxicol.

Environ. Int.

Environ. Pollut.

Aquat. Toxicol.

Sci. Total Environ.

Comp. Biochem. Physiol. C

Utility of tissue residues for predicting effects of metals on aquatic organisms

Integr. Environ. Assess. Manag.

Biochemical and morphological responses in Chironomus riparius (diptera, chironomidae) larvae exposed to lead-spiked sediment

Environ. Toxicol. Chem.

Effects of mercury on growth, emergence and behavior of Chironomus riparius meigen (diptera: chrironomidae)

Arch. Environ. Contam. Toxicol.