Dirk Dobritzsch¹
email: dirk.dobritzsch@biochemtech.uni-halle.de

Konstantin Grancharov²

Corinna Hermsen¹

Gerd-Joachim Krauss¹

Dirk Schaumlöffel

Inhibitory effect of metals on animal and plant glutathione transferases #

Glutathione transferases (GSTs) represent a widespread enzyme superfamily in eukaryotes and prokaryotes catalyzing different reactions with endogenous and xenobiotic substrates such as organic pollutants. The latter are often found together with metal contamination in the environment. Besides performing of essential functions, GSTs protect cells by conjugation of glutathione with various reactive electrophiles. The interference of toxic metals with this functionality of GSTs may have unpredictable toxicological consequences for the organisms. In this review results from the recent literature are summarized and discussed describing the ability of metals to inhibit intracellular detoxification processes in animals and plants.

Introduction

Metal pollutants are one of the greatest challenges to the environment and human health. Metals enter the biosphere by natural processes (e.g. volcanism, weathering) and human activities such as mining, fuel combustion and many other industrial processes [START_REF] Garrett | Natural sources of metals to the environment[END_REF]. The toxicological relevance of metals depends on their chemical species, i.e. the chemical compound and the oxidation state of the metal, as well as the bioavailability of these metal species for organisms. In the environment biogeochemical transformations of metal species have to be taken into account. For example in aquatic systems the inorganic mercury species Hg 2+ can be transformed by microorganisms into highly toxic methylmercury (MeHg) that represents the most environmental concern regarding this element [START_REF] Benoit | Geochemical and biological controls over methylmercury production and degradation in aquatic ecosystems[END_REF]. Another example are acid mine drainage waters which are acidic effluents from abandoned mines which can contain very high amounts of arsenic. Biogeochemical processes involving microorganisms oxidize arsenite (AsO3 3-) to arsenate (AsO4 3-) together

Characteristics of glutathione transferase and its role in biological systems

The glutathione transferase (formerly reported as glutathione S-transferases, EC 2.5.1.18) supergene family is composed of several isoenzymes which are involved in phase II detoxification of electrophilic xenobiotics and endogenous compounds in procaryotes [START_REF] Allocati | Glutathione transferases in bacteria[END_REF][START_REF] Allocati | Distribution of glutathione transferases in Gram-positive bacteria and Archaea[END_REF] and eucaryotes including non-mammalian vertebrates [START_REF] Sheehan | Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily[END_REF], mammals and humans [START_REF] Dourado | Mammalian cytosolic glutathione transferases[END_REF][START_REF] Jancova | Phase II drug metabolizing enzymes[END_REF] and plants [START_REF] Dixon | Roles for glutathione transferases in plant secondary metabolism[END_REF][START_REF] Cummins | Multiple roles for plant glutathione transferases in xenobiotic detoxification[END_REF][START_REF] Kumar | Glutathione S-Transferases: Role in Combating Abiotic Stresses Including Arsenic Detoxification in Plants[END_REF]. GSTs catalyze the conjugation of organic compounds to the nucleophilic sulfur of the major intracellular thiol compound, glutathione (GSH) rendering them more hydrophilic and less toxic.

Recently, a graphical overview of the different classes of glutathione transferase was proposed by Kumar and Trivedi [16] (Figure 2). At least 16 genes encode GSTs in the mammalian cytosol, whereas for plants a number of 40-60 is proposed [START_REF] Frova | Glutathione transferases in the genomics era: New insights and perspectives[END_REF]. They are mostly dimeric enzymes of 45-55 kDa and include eleven classes of mammalian cytosolic GSTs: A (alpha), M (mu), P (pie), C (chie), D (delta), B( beta), E (epsilon), O (omega), S (sigma), T (theta), and Z (zeta), both latter classes are found in plants, too . To date six classes of plant cytosolic GSTs have been identified Z (zeta), T (theta), F (phi), U (tau), L (lambda), and DHAR (dehydroascorbate reductase)) [START_REF] Frova | Glutathione transferases in the genomics era: New insights and perspectives[END_REF][START_REF] Edwards | Compartmentation of photosynthesis in cells and tissues of C-4 plants[END_REF]. Except for L (lambda) and DHAR (dehydroascorbate reductase), which are monomers, plant GSTs also occur as dimers. In plants phi and tau class GSTs catalyze peroxidase reactions, while zeta class GSTs catalyze isomerase reactions [START_REF] Dixon | Plant glutathione transferases[END_REF]. In each class, multiple isoenzymes were found whereas tandem duplication can be regarded as the major expansion mechanism [START_REF] Chi | Expansion Mechanisms and Functional Divergence of the Glutathione S-Transferase Family in Sorghum and Other Higher Plants[END_REF].

In mammals, cell specific distribution of GST isoforms has been reported, as GSTP in the biliary tract and GSTA in hepatocytes [START_REF] Hayes | Glutathione Stransferases in man: the relationship between rat and human enzymes[END_REF]. A high expression of cytosolic GST P, M, A, and T classes in various tumor cells has been observed [START_REF] Tew | Glutathione-associated Enzymes in Anticancer Drug Resistance[END_REF][START_REF] Townsend | The role of glutathione-S-transferase in anti-cancer drug resistance[END_REF]. Typical GST-catalyzed reactions include: i) nucleophilic aromatic substitution, ii) Michaeltype addition, iii) nucleophilic addition to epoxides, iv) hydroperoxide reduction, and v) double bond isomerization [START_REF] Johansson | Human glutathione transferase A3-3, a highly efficient catalyst of double-bond isomerization in the biosynthetic pathway of steroid hormones[END_REF].

Besides detoxification, GSTs perform other essential functions in animal and plant cells [START_REF] Board | Glutathione transferases, regulators of cellular metabolism and physiology[END_REF][START_REF] Nianiou-Obeidat | Plant glutathione transferase-mediated stress tolerance: functions and biotechnological applications[END_REF]. GSTs have regulatory roles in cellular signaling (e.g. by mediating S-glutathionylation of specific clusters of target proteins and in reactions that define a negative regulatory role in some kinase pathways through ligand or protein:protein interactions, example for the role of GSTs´ in cellular signaling [START_REF] Tew | The role of glutathione S-transferase P in signaling pathways and S-glutathionylation in cancer[END_REF] and inactivate endogenous products of oxidative stress (such as 13-oxooctadecadienoic acid and 4-hydroxynonenal). These compounds are known to elicit cellular responses, such as apoptosis and cell proliferation. Conjugation with GSH eliminates these effects [START_REF] Hubatsch | Human glutathione transferase A4-4: an Alpha class enzyme with high catalytic efficiency in the conjugation of 4-hydroxynonenal and other genotoxic products of lipid peroxidation[END_REF][START_REF] Sharma | Transfection with 4-hydroxynonenal-metabolizing glutathione S-transferase isozymes leads to phenotypic transformation and immortalization of adherent cells[END_REF].

GSTs also have non-catalytic functions. Originally, GSTs were identified as "ligandins" due to their ability to bind and transport across the plasma membrane organic molecules that are not enzyme substrates [START_REF] Tipping | Binding of porphyrins by ligandin[END_REF]; e.g. steroids, thyroid hormones, bile acids, bilirubin and heme [START_REF] Danielson | Kinetic independence of the subunits of cytosolic glutathione transferase from the rat[END_REF]. This however might just be some sort of cross reaction due to the chemical abilities rather than a specific, regulated function of GSTs.

Plant GSTs are active as hormone and flavonoid ligandins [START_REF] Nianiou-Obeidat | Plant glutathione transferase-mediated stress tolerance: functions and biotechnological applications[END_REF][START_REF] Mueller | AN9, a petunia glutathione Stransferase required for anthocyanin sequestration, is a flavonoid-binding protein[END_REF]. Tau-type GSTs are able to bind GSH conjugates of fatty acids [START_REF] Dixon | Selective Binding of Glutathione Conjugates of Fatty Acid Derivatives by Plant Glutathione Transferases[END_REF].

Probably, plant GSTs are involved in in vivo synthesis of sulfur-containing secondary metabolites such as volatiles and glucosinolates as well as in conjugation, transport and storage of reactive oxylipins, phenolics and flavonoids [START_REF] Dixon | Roles for glutathione transferases in plant secondary metabolism[END_REF][START_REF] Cummins | Multiple roles for plant glutathione transferases in xenobiotic detoxification[END_REF][START_REF] Hasanuzzaman | Glutathione in plants: biosynthesis and physiological role in environmental stress tolerance[END_REF]. During plant developmental processes and stress response GSTs play also an important role in the regulation of cellular detoxification processes under different light conditions [START_REF] Galle | Plant Glutathione Transferases and Light[END_REF]. Gullner et al. [START_REF] Gullner | Glutathione S-Transferase Enzymes in Plant-Pathogen Interactions[END_REF] summarize diverse roles of plant GST isoenzymes in the interaction of plants with pathogenic bacteria and fungi.

In mammalian systems, GSTs participate in cell signaling through protein-protein interactions with many regulatory signaling proteins, such as c-Jun N-terminal kinase (JNK) and apoptosis signal regulating kinase ASK1 [START_REF] Edalat | Selective recognition of peptide sequences by glutathione transferases: A possible mechanism for modulation of cellular stress-induced signaling pathways[END_REF]. Activation of these kinases leads either to apoptosis or cellular proliferation [START_REF] Liu | Thioredoxin promotes ASK1 ubiquitination and degradation to inhibit ASK1-mediated apoptosis in a redox activity-independent manner[END_REF]. GSTs can inhibit JNK and ASK1 activity by forming complexes [START_REF] Townsend | The role of glutathione-S-transferase in anti-cancer drug resistance[END_REF][START_REF] Monaco | Identification of a glutathione-S-transferase effector domain for inhibition of jun kinase, by molecular dynamics[END_REF]. Thus, GSTs play a decisive cytoprotective role against carcinogenic electrophiles and products of oxidative stress. Perturbation of their function may have unpredictable toxicological consequences. Previously the concept of GST inhibition in ameliorating anticancer drug resistance was reviewed [START_REF] Ruzza | Glutathione Transferases as Targets for Cancer Therapy[END_REF][START_REF] Sau | Glutathione transferases and development of new principles to overcome drug resistance[END_REF]. In recent years novel functions of GSTs have been suggested such as regulation of cell signaling pathways and ion channels [START_REF] Board | Glutathione transferases, regulators of cellular metabolism and physiology[END_REF].

Inhibition of GST by toxic metals in man, animals and plants

A principal function of the GST enzymes is the detoxification of xenobiotic substances by catalysing their conjugation with glutathione. This GST activity can be interfered by metals which may lead to detrimental toxic effects for the organism (Figure 1). Data on in vivo and in vitro inhibitory effects of metal on GSTs are summarized in Table 1. In most of the studies the influence of toxic metals or surplus essential biometals to total GST activity was determined by measuring the absorbance (340 nm) of the conjugation of the model xenobiotic 1-chloro-2,4-dinitrobenzene (CDNB) with reduced GSH (Figure 3), usually according to a method published by Habig et al. [START_REF] Habig | Glutathione S-Transferases -First Enzymatic Step in Mercapturic Acid Formation[END_REF]. The increase of absorbance is directly related to the GST activity. The method shows a good reproducibility, when summarizing the different studies reviewed here. The overall standard deviation for GST activity determination was around 3% in most of the studies. In the following sections the metals are listed according to Periodic Table of the Elements with increasing average atomic mass from light to heavy.

GST inhibition in animals including men

Although most studies have been conducted with laboratory animals or fish, here we report published effects that will obviously occur in all types of animals, including humans. Although the qualitative and quantitative expression of the effects may vary greatly according to the type of organism and its physiological state, as well as the type of metals and mode of application. Aluminium Aluminium can be found ubiquitously in the environment either naturally or introduced due to human activity. This metal has no known essential biological function. However, it has the potential to cause neurotoxic effects in man and animals. For example, Al has been connected to neurodegenerative diseases such as Alzheimer's and Parkinson's disease, as well as amyotrophic lateral sclerosis. Therefore, the few studies describing Al influence on GST activity have been mainly done in brain tissue. A recent study of Singla and Dhawan [START_REF] Singla | Zinc modulates aluminium-induced oxidative stress and cellular injury in rat brain[END_REF] detected a highly significant decrease of GST activity by 68% in cerebellum and to 41% in cerebrum of male Sprague Dawley rats after oral administration of AlCl3 at a daily dose of 100 mg kg -1 for a total duration of 8 weeks. Unfortunately, the used GST conjugate was not mentioned which limits the comparability with other studies. However, these results confirmed an earlier study of Sharma et al. [START_REF] Sharma | Role of combined administration of Tiron and glutathione against aluminum-induced oxidative stress in rat brain[END_REF] who measured a decrease of GST activity (CDNB conjugate) by 24% in brains of female Wistar rats after oral administration of AlCl3 (172.5 mg kg -1 day -1 ). Even at lower doses of AlCl3 (orally 34 mg kg -1 day -1 ) a decrease of GST activity (p-nitrobenzylchloride conjugate) to 19-24% was detected in plasma, liver, kidney, testes but not brain of male Sprague Dawley rats [START_REF] El-Demerdash | Antioxidant effect of vitamin E and selenium on lipid peroxidation, enzyme activities and biochemical parameters in rats exposed to aluminium[END_REF].

Chromium

Cr III was considered to be required as a trace element in metabolism [START_REF] Mertz | Chromium in human nutrition -a review[END_REF] but results of new studies indicate that chromium currently can only be considered pharmacologically active with beneficial effects have not been definitively shown to occur in animals [START_REF] Vincent | New Evidence against Chromium as an Essential Trace Element[END_REF]. Hexavalent chromium easily enters the cell where it is reduced to trivalent chromium compounds by thiols and cytochrome P450-dependent monooxygenases (P450-MO) [START_REF] Gerhardsson | Concepts on biological markers and biomonitoring for metal toxicity[END_REF]. Cr VI is very toxic and mutagenic [START_REF]Toxicological Profile: Chromium, The Agency for Toxic Substances and Disease Registry (ATSDR)[END_REF]. The coordinated regulation and expression of both enzyme systems GST and P450-MO could result in diminished detoxification capacity of the organism, and severe toxicity of Cr VI compounds. Exposure of the frog Rana ridibunda to chromium (10 ppm K2Cr2O7, 14 days) caused decreased liver (58%) and kidney (56%) GST activities [START_REF] Kostaropoulos | Effects of exposure to a mixture of cadmium and chromium on detoxification enzyme (GST, P450-MO) activities in the frog Rana ridibunda[END_REF]. Similar decline has been also reported for P450-MO.

Copper

In low concentrations copper is an essential micronutrient. Copper-containing proteins play a role in energy production, signalling and proliferation. Copper deficiency or excess can lead to acute or chronic toxicity triggered by redox-dependent chelation and regulatory mechanisms. For humans, the toxicity limit is more than 10 μM, [START_REF] Georgopoulos | Environmental copper: Its dynamics and human exposure issues[END_REF][START_REF]National_Research_Council, Copper in Drinking Water[END_REF]. Excess bioavailable copper thus acts as a genotoxic pollutant and leads to oxidative stress, DNA and protein damage. CuSO4 significantly inhibited GST activity in the gill homogenate of Oncorhynchus mykiss (rainbow trout, [START_REF] Arabi | Metal-ion-mediated oxidative stress in the gill homogenate of rainbow trout (Oncorhynchus mykiss) -Antioxidant potential of manganese, selenium, and albumin[END_REF] and as recently shown in the larvae of the insect Protaetia brevitarsis which play an important role for composting and rotting of fungal and plant residues [START_REF] Yin | Biochemical responses of the Protaetia brevitarsis Lewis larvae to subchronic copper exposure[END_REF]. In the latter case, firstly an increase of GST activity was observed after 7 days of Cu exposure followed by a significant decrease after 28 d. Letelier et al. [START_REF] Letelier | Aracena-Parks, Inhibition of cytosolic glutathione S-transferase activity from rat liver by copper[END_REF] reported that Cu 2+ either alone or in the presence of ascorbate (ROS generating system) produced dose-and time-dependent inactivation of GST in rat liver cytosol. Copper significantly modified GST affinity to GSH, (Vmax ↓, Km ↑) while substrate affinity for xenobiotics remained unchanged. The major mechanism of Cu 2+ -induced GST inhibition is evidenced to oxidation of its thiol groups and not direct binding to thiol groups.

Arsenic

Arsenic is one of the most harmful metalloids in the environment. Chronic exposure to AsO3 3-is associated with oxidative stress and increased risk of cancer (skin, bladder, and lung). Swiss mice exposed to arsenic (NaAsO2, 10 mg × kg -¹, 2 days, orally) showed accumulation in cardiac and renal tissues, accompanied with significant reduction of antioxidant enzyme activities [START_REF] Manna | Arsenic-induced oxidative myocardial injury: protective role of arjunolic acid[END_REF][START_REF] Sinha | Arjunolic acid attenuates arsenic-induced nephrotoxicity[END_REF]. Also, cardiac (53%) and renal (63%) GST activities were reduced. Chronic exposure of C57B16 mice to low As levels (50 ppb, NaAsO2, 4 weeks) fully blocked GST-ω expression in the lung-lining fluid [START_REF] Lantz | Pulmonary biomarkers based on alterations in protein expression after exposure to arsenic[END_REF]. Arsenic at low concentrations (500 µg × L -¹ of As2O3, 7 days) interfered with antioxidant system of the polychaete Laeonereis acuta. Thus, 48% lower conjugation capacity of GST compared to controls was observed [START_REF] Ventura-Lima | Toxicological responses in Laeonereis acuta (annelida, polychaeta) after arsenic exposure[END_REF].

Antimony

In the environment natural concentration of the toxic metalloid antimony is low. However, Sb may occur on higher levels at industrial sites such as mining and processing which raises concerns about human and environmental exposure [START_REF] Tamas | Cellular and molecular mechanisms of antimony transport, toxicity and resistance[END_REF]. Among a series of metals tested for their effects toward GST from human erythrocytes, trivalent antimony (Sb³ + , antimony potassium tartrate) was found to be the most efficient inhibitor (IC50: 50 µM), followed by Hg² + , Cu² + , Cd² + , Cr³ + and Fe² + [START_REF] Poon | Effects of trivalent antimony on human erythrocyte glutathione-Stransferases[END_REF]. While Sb³ + was a competitive inhibitor towards CDNB (Ki: 18 µM), evidenced by interaction of Sb³ + with GSH, Sb 5+ failed to exert any effect on GST.

Selenium

Selenium is an essential trace element but at high concentrations it becomes toxic. For example, Se toxicity to aquatic organisms has been studied with respect to the GST activity by Gobi et al. [START_REF] Gobi | Bioaccumulation, cytotoxicity and oxidative stress of the acute exposure selenium in Oreochromis mossambicus[END_REF] in gill and liver cytosols of the fish Oreochromis mossambicus native to southern Africa. In liver GST activity decreased about 20% when exposed to 100 µg/L Se in comparison to water only (control) for periods of 96 h. Interestingly, at the same Se concentration the GST activity in gill increased by approximately 30%. One conceivable hypothesis is that Se supplementation leading to an increase of GPXs activities which may enable a decrease of GST activity / GSH synthesis to minimize efforts for antioxidant capacity.

Tellurium

The influence of tellurium at toxic concentrations on GST activity in animal tissue has been rarely investigated. Tellurium, a rare element of raising importance, occurs in dusts from blasts furnace refining of lead. Given to mice (orally, 15 days, 2 mg × kg -¹, sodium salt), it caused dose-dependent decrease of activity of GSH and antioxidant enzymes as well as reduced GST activity in cerebrum (37%), cerebellum (26%) and brain stem (29%) [START_REF] Kaur | Tellurium-induced dosedependent impairment of antioxidant status -Differential effects in cerebrum, cerebellum, and brainstem of mice[END_REF].

Silver

Silver levels are high in mineral rich areas and polluted soils. The monovalent silver ions reduce cell viability. In all inhibitory studies referred here, Ag + shows the highest inhibitory effect to GSTs. Using purified rainbow trout erythrocytes the in vitro effects of various metal ions on GST activities were measured [START_REF] Comakli | Effects of some metal ions on rainbow trout erythrocytes glutathione S-transferase enzyme: an in vitro study[END_REF]. Inhibition constant (Ki values) and IC50 values (concentration of inhibitor necessary to halve the reaction rate of an enzyme-catalyzed reaction) demonstrate that Ag + show the highest inhibitory effect. But, Cd 2+ and Cr 2+ inhibited the GST strongly, too (Tab. 1). GST in muscle tissue from Van Lake fish (Chalcalburnus tarichii) was inhibited mostly strong in a non-competitive manner by Ag + , but also by other metals (Tab.1). Cd 2+ , Co 2+ and Fe 2+ caused no inhibitory effect [START_REF] Aksoy | Purification of glutathione S-transferase from Van Lake fish (Chalcalburnus tarichii Pallas) muscle and investigation of some metal ions effect on enzyme activity[END_REF]. Similar results are shown for Van Lake fish gills [START_REF] Ozaslan | Some metals inhibit the glutathione Stransferase from Van Lake fish gills[END_REF]. Highly purified GST from turkey liver is inhibited in the following order: Ag + > Cu 2+ > Hg 2+ [START_REF] Akkemik | Purification and characterization of glutathione S-transferase from turkey liver and inhibition effects of some metal ions on enzyme activity[END_REF].

Within the last decades the use of nanomaterials in various products has increased. Among different nanomaterials, silver nanoparticles (AgNPs) are extensively used in many commercial products [START_REF] Maynard | Safe handling of nanotechnology[END_REF]. Thus, AgNPs will reach the environment and cause effects at various levels in aquatic organisms [START_REF] Asharani | Toxicity of silver nanoparticles in zebrafish models[END_REF] or soil organisms [START_REF] Sillapawattana | Effect of silver nanoparticles on the standard soil arthropod Folsomia candida (Collembola) and the eukaryote model organism Saccharomyces cerevisiae[END_REF]. The induction of GSTs mRNA triggered by Cd and AgNPs seems to show that GST are important for defense in aquatic organisms [START_REF] Nair | Identification, characterization and expression profiles of Chironomus riparius glutathione S-transferase (GST) genes in response to cadmium and silver nanoparticles exposure[END_REF]. However, change of GST activity was not significant with increasing concentration of AgNPs in Folsomia candida (Collembola) but effects of AgNPs is similar to the one from AgNO3 [START_REF] Sillapawattana | Effect of silver nanoparticles on the standard soil arthropod Folsomia candida (Collembola) and the eukaryote model organism Saccharomyces cerevisiae[END_REF].

Cadmium

Cadmium, one of the most hazardous pollutants, can be accumulated in aquatic organisms. The gills of fishes are major targets for acute metal toxicity in these organisms due to their permanent contact with water contaminants. Long term (2 weeks) low-level (2 µg × L -¹) exposure to Cd² + led to a distinct decrease of GST activity in gills (> 80%) , but also in digestive glands (> 60%) of the freshwater crayfish Astacus astacus [START_REF] Meyer | Biochemical and histochemical observations on effects of low-level heavy metal load (lead, cadmium) in different organ systems of the freshwater crayfish, Astacus astacus L. (Crustacea: Decapoda), Ecotox[END_REF]. In Cd 2+ exposed freshwater crab (Sinopotamon yangtsekiense) GSH and antioxidant enzymes activities were gradually reduced in gills with increasing concentrations (7-116 mg × L -¹) and duration [START_REF] Wang | Effects of cadmium on glutathione synthesis in hepatopancreas of freshwater crab, Sinopotamon yangtsekiense[END_REF]. GST activity was decreased by 44% after exposure to 116 mg× L -¹ Cd 2+ for 96 h.

In mice liver GST showed a cadmium (up to 2.5 mg × L -¹ CdCl2) -induced, duration (up to 7 alternate days)dependent depression of conjugating capacity (35%) [START_REF] Karmakar | Cadmium-induced alterations of hepatic lipid peroxidation, glutathione S-transferase activity and reduced glutathione level and their possible correlation with chromosomal aberration in mice: a time course study[END_REF]. This was correlated with elevated hepatic lipid peroxidation (102%) and chromosomal aberrations (30%). CdCl2 (4 mg × kg -¹) also caused organ specific reduction of GST activity in heart (53%) and renal (45%) tissues of mice upon 6 or 3 days [START_REF] Manna | Amelioration of cadmium-induced cardiac impairment by taurine[END_REF]. Concomitant accumulation of Cd in these tissues increased lipid peroxidation products and protein carbonyl contents, as well as decreased activities of other antioxidant enzymes. Murine hepatocytes, treated with 800 µM CdCl2 for 3 h showed reduced GST activity associated with significant reduction of cell viability, increased levels of lipid peroxidation products and oxidized glutathione [START_REF] Sinha | Attenuation of cadmium chloride induced cytotoxicity in murine hepatocytes by a protein isolated from the leaves of the herb Cajanus indicus L[END_REF].

Tin

Organotin compounds such as tributyltin (TBT), triphenyltin (TPT) and dibutyltin (DBT), are extensively used as plastic stabilizers, wood preservatives and antifouling agents in paints. Due to their widespread use, high amounts of butyltins have polluted the environment causing harmful toxic effects. Fishes are suitable organisms to evaluate the effect of such contaminants in aquatic ecosystems [START_REF] Fang | The importance of environmental factors and matrices in the adsorption, desorption, and toxicity of butyltins: a review[END_REF]. Organotins strongly inhibit GST activities in marine fish liver and kidney cytosol showing a 10 (liver) to 100 (kidney) times higher inhibition compared to inorganic tin [START_REF] Al-Ghais | Inhibition of glutathione-S-Transferase catalyzed xenobiotic detoxification by organotin compounds intropical marine fish tissues[END_REF]. This highlights the fact that inhibition depends on the chemical species. IC50 values for hepatic GST ranged between 7.4-18 µM for Siganus canaliculatus (Rabbitfish), and between 17-45 µM for Sparus sarba (Arabian seabream). The order of inhibitory potency was TBT > TPT > DBT; reversible inhibition was observed in all cases. Several times lower IC50 values have been recorded for kidney GST (0.92-4 µM) indicating significantly higher sensitivity of renal isoenzymes to organotins. The results were consistent with previous in vitro competitive and reversible inhibition of rat liver GSTs by TBT and TPT [START_REF] Henry | Inhibition of glutathione-S-aryltransferase from rat liver by organogermanium, lead and tin compounds[END_REF]. Organotin compounds are a special case when studying GST inhibition as they represent metals and organic xenobiotics in the same time. All studies proposed an inhibitory effect via a direct binding of the organotins to the active site of GST. Recent in vivo studies on TBT towards hepatic GST in Arctic charr (Salvelinus alpinus, 0.3 mg × kg -¹, at long-term exposure) confirmed the potent inhibitory effect but discussed also the possibility that organotins may compete with the detoxification of other organic xenobiotics for binding to GST and GSH [START_REF] Padros | Metabolic interactions between low doses of benzo a pyrene and tributyltin in arctic charr (Salvelinus alpinus): a long-term in vivo study[END_REF].

Mercury

Mercury is present in the environment as several different chemical species showing a range of toxicological properties. These chemical forms can be classified as elemental (Hg 0 ), inorganic (e.g. Hg 2+ ), and organic mercury, mainly alkyl mercury such as methyl mercury (MeHg). Goodrich and Basu [START_REF] Goodrich | Variants of glutathione s-transferase pi 1 exhibit differential enzymatic activity and inhibition by heavy metals[END_REF] proved the influence of mercury among many other metals to genetically engineered GST allozymes. All allozymes were inhibited strongly by Hg 2+ (but also MeHg and selenium) (Tab.1). In a comprehensive in vitro study Sen and Semiz [START_REF] Sen | Effects of metals and detergents on biotransformation and detoxification enzymes of leaping mullet (Liza saliens)[END_REF] investigated the effects of mercury and other toxic metals on cytosolic liver GST activities in the fish leaping mullet (Liza saliens). Both CDNB-and EA-GST were mainly inhibited by Hg² + (CDNB-GST 70% and EA-GST 86%, resp.), but also by Sb² + (69% and 78%), and Cd² + (65% and 69%). Other tested metals Cu² + , Zn² + , Co² + , Fe 3+ , and Fe 2+ exhibited more moderate effects (41-61%). The fail of fully reactivation by GSH pointed to inhibition mechanisms other than simple binding to SH-groups by toxic metals. In a recent study of Gunderson et al. [START_REF] Gunderson | Response of phase I and II detoxification enzymes, glutathione, metallothionein and acetylcholine esterase to mercury and dimethoate in signal crayfish (Pacifastacus leniusculus)[END_REF] in the signal crayfish Pacifastacus leniusculus a similar decrease of the GST activity in hepatopancreas cytosol was observed under Hg 2+ exposition at concentrations comparable to previous studies.

Lead

A continuous, low-level exposure of the freshwater crayfish Astacus astacus to lead (20 µg × L -¹) resulted in strong decrease of GST activity in gills and digestive gland [START_REF] Meyer | Biochemical and histochemical observations on effects of low-level heavy metal load (lead, cadmium) in different organ systems of the freshwater crayfish, Astacus astacus L. (Crustacea: Decapoda), Ecotox[END_REF]. Accordingly, increased metal accumulation and severe structural impairment in these organs were observed. Thus, a chronic low-level metal stress may have a biological impact as great as short-term acute dose insults in aquatic ecosystems. Long term low-level lead exposure (0.1% in drinking water) of rats has been reported to profoundly impair phase I and phase II metabolising enzyme activity in the lenticular system of the animals. A very pronounced decrease (up to 55% inhibition) was noticed in GST activity [START_REF] Dwivedi | Lead exposure alters the drug metabolic activity and the homeostasis of essential metal ions in the lenticular system of the rat[END_REF].

Cd 2+ and Pb 2+ have been shown to be potent inhibitors (by more than 50% at 100 µM) of pulmonary GST in rabbit lung cytosol [START_REF] Nakahama | Comparative study on in vitro inhibitory effects of heavy metals on rabbit drug-metabolizing enzymes[END_REF]. Since GST and other detoxifying enzymes were found to be extremely poor in lung, in vitro enzyme assays are suitable tools to indicate pulmonary toxicities.

GST inhibition in plants

Trace elements such as Cu, Zn, Mn, Fe, Mo and Ni are essential in small amounts for plant metabolism while higher concentrations can cause severe toxic effects [START_REF] Hansch | Physiological functions of mineral micronutrients (Cu[END_REF]. Only few studies investigated the inhibition of GST activity in plants by metals such as Cu, Zn, Cd, As, Pb, and Ni. Recently, Kumar and Trivedi [16] gave an overview regarding the role of GST for arsenic detoxification in plants.

Copper

Due to its redox activity (Cu + , Cu² + ) copper is of high significance for plants (respiratory chain: cytochrome oxidase, photosynthesis: plastocyanine). On the other hand, copper induce reactive oxygen species (ROS) by autoxidation and Fenton [START_REF] Jonak | Heavy metal stress. Activation of distinct mitogen-activated protein kinase pathways by copper and cadmium[END_REF]. Ahsam et al. [START_REF] Ahsan | Excess copper induced physiological and proteomic changes in germinating rice seeds[END_REF] did a general study of Cu² + influence in rice (Oryza sativa) germination. During proteomic research in the first 4 days of germination, GST (identified by peptide-mass fingerprinting) showed a slightly decrease upon CuSO4 exposure (1.5 mM, 4 d). Unfortunately the authors did not assay enzyme activities. However, in a recent study Li et al. [START_REF] Li | Glutathione S-transferases modulate Cu tolerance in Oryza sativa[END_REF] measured the GST activity in roots of two varieties of Oryza sativa seedlings. In the Cu sensitive variety GST activity decreased by about 52% after 3 days Cu exposure (8 µM) while the Cu tolerant variety showed only a GST activity decrease of 30%. In duckweed (Lemna minor) GST activity was inhibited time-and concentration-dependently by CuSO4 [START_REF] Teisseire | Copper-induced changes in antioxidant enzymes activities in fronds of duckweed (Lemna minor)[END_REF]. For example, low Cu exposure (0.25 μM to 0.5 μM) lad to a stimulation (114.4% to 125%) of GST as a consequence of CuSO4 addition. While higher Cu content (2 μM) reduced GST activity (60% of the control level).

Zinc

Zinc is typically the second (after iron) most abundant transition metal in plants [START_REF] Broadley | Zinc in plants[END_REF]. In poplar lines different effects of ZnSO4 for GST activity were measured [START_REF] Bittsanszky | Ability of transgenic poplars with elevated glutathione content to tolerate zinc(2+) stress[END_REF]. Leaf disks of 2 hybrids (Populus nigra (N-SL clone), P. canescens (P. termula  P. alba)) and 2 transgenic (P. canescens) clones, overexpressing E. coli γ-ECS (11ggs: cytosol, 6LgI: chloroplast)) were exposed to Zn (10 -5 -10 -1 M, 21 days). In clone 11ggs GST activity was consistently lowered by 10 -5 -10 -1 M ZnSO4. In both hybrids GSTs were inhibited at high (cytotoxic) Zn 2+ concentrations (10 -2 and 10 -1 M).

Cadmium, arsenic, lead and nickel

Cadmium which chemically is similar to zinc, generally causes toxic effects in plant species. Interest in Cd tolerance mechanisms [START_REF] Ernst | Interaction of heavy metals with the sulphur metabolism in angiosperms from an ecological point of view[END_REF][START_REF] Rother | Sulphate assimilation under Cd2+ stress in Physcomitrella patens -combined transcript, enzyme and metabolite profiling[END_REF] is often driven by potential applications in phytoremediation [START_REF] Lyubenova | Plants for waste water treatment -Effects of heavy metals on the detoxification system of Typha latifolia[END_REF][START_REF] Memon | Implications of metal accumulation mechanisms to phytoremediation[END_REF]. While GST activities in 25 day old hydroponic cultures of Indian mustard (Brassica juncea L. cv. Vitasso) after Cd 2+ (5 days, 10 µM) which is environmentally relevant, were not influenced, higher metal concentrations decreased GST activity significantly to 61.3% (30 µM), 82.4% (50 µM) and 66.4% (100 µM) [START_REF] Markovska | Cadmium-induced oxidative damage and antioxidant responses in Brassica juncea plants[END_REF]. Interestingly no continuous decrease was observed. A 10 days exposure of Cd (40 mg L-1) to rice (Oryza sativa) showed a significant inhibition of GST activity by 63% in rice roots in contrast to a minimal increase in shoots [START_REF] Zhang | Response of Glutathione and Glutathione S-transferase in Rice Seedlings Exposed to Cadmium Stress[END_REF].

Typha latifolia is some of the potent wetland plants for phytoremediation. It is used in constructed wetlands, an alternative to traditional industrial wastewater treatment [START_REF] Guittonny-Philippe | Constructed wetlands to reduce metal pollution from industrial catchments in aquatic Mediterranean ecosystems: A review to overcome obstacles and suggest potential solutions[END_REF]. After 72h exposures of Typha latifolia either to Pb, arsenite, or Cd showed remarkable GST inhibitions in leaves [START_REF] Lyubenova | Plants for waste water treatment -Effects of heavy metals on the detoxification system of Typha latifolia[END_REF].

Arsenic and lead are taken up by plants from the soil, water or air. Spruce cell culture (Picea abies L.Karst.) were exposed for 16 h to 50-500 µM CdSO4, 1.5-80 µM Na2HAsO4 and 50-400 µM PbCl2 [START_REF] Schröder | Reaction of detoxification mechanisms in suspension cultured spruce cells (Picea abies L. Karst.) to heavy metals in pure mixture and in soil eluates[END_REF]. To distinguish between different GST isoforms 4 different standard substrates for activity assay were used. Only assays with 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP) as substrate showed remarkable inhibition effects (60%) caused by cadmium. Under lead exposure the same effect was observed with a slightly higher residual enzyme activity (56%). Arsenic inhibited EPNP conjugation continuous with higher concentration. GST was assayed in horseradish (Armoracia rusticana L.) hairy root culture, incubated for 6 h with 0.1 and 0.5 mM Cd 2+ , Ni 2+ and Pb 2+ using 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) in order to detect different GST isoforms [START_REF] Nepovím | Effects of heavy metals and nitroaromatic compounds on horseradish glutathione S-transferase and peroxidase[END_REF]. While GST activity with CDNB was not influenced, assays with DCNB showed decreasing activity levels in a metal dependent manner in the range of 35% (for Ni 2+ ) to 63% (for Cd 2+ ).

Conclusions

Metals represent a major ecological concern because they can accumulate in organisms in the food web and finally, they can be a threat to human health. Impact of metals to aquatic and terrestrial ecosystems at sublethal contaminant concentrations can cause biological responses of the glutathione antioxidative network including glutathione transferases in organisms living in polluted habitats. On cellular level, metals are able to inhibit different intracellular processes such as enzyme activities. Inhibition of GSTs in animals and plants represents an early warning biomarker indicating a sensitive physiological response of organism to metals. In this context the measurement of GST activity is an important tool. In most studies, metals decreased GST activity supporting the fact that GST is part of the cellular antioxidant defense system where it is serving as bifunctional enzyme: GST and as glutathione peroxidase, too. Furthermore, animal and plant glutathione transferases are involved in detoxification of xenobiotics and endobiotic metabolites. Therefore, interferences with the functionality of GSTs caused by toxic metals or imbalanced essential biometals may have unpredictable toxicological consequences for the organisms. However, it is difficult to make a general assessment of the level at which natural exposure, dietary supplementation, over-nutritional dietary supplementation or poisoning will show effects.

When reviewing the literature on animal studies it can be concluded that the majority of articles are dealing with organisms living in aquatic ecosystems. Here, fishes are of particular interest because in contaminated aquatic environments they are in direct contact with toxic substances in contrast to terrestrial animals which have a rather selective contact with toxic substances in a contaminated site. Fish gills are organs and tissue which are primarily affected because they provide a large surface area in contact with contaminants dissolved in water. Gill cells respond rapidly to metals and thus gills are major targets for measurement of acute toxicity via GST activity. Moreover, some fish species such as signal crayfish are living in relatively small habitats; therefore they can serve as good bioindicators for specific exposed areas.

Rather few studies have investigated the impact of metals to GST activity in plants although plants as sessile organisms represent suitable bioindicators for specific contaminated sites as well as for long-term observation of pollution. With respect to metal polluted aquatic ecosystems only one plant study has been published so far. It investigated the aquatic macrophyte Typha latifolia which represents a common wetland plant used in some cases for phytoremediation in constructed wetlands for sustainable waste water treatment. This plant can accumulate metals, however, after long-term exposure a decrease of GST activity was observed. This particular case demonstrates that there is a lack of studies in this field. In future, more investigations on GST activity in plants exposed to toxic metal in aquatic as well as in terrestrial ecosystems would be desirable in order to fill this gap of knowledge. * Membrane-associated glutathione S-transferases not involved in intracellular detoxification processes were not considered here. These GSTs are more likely to be involved in special developmental processes (e.g. heat shock in stationary phase and ability to grow at 39 °C, [START_REF] Choi | A novel membrane-bound glutathione S-transferase functions in the stationary phase of the yeast Saccharomyces cerevisiae[END_REF]. Membrane bound microsomal GSTs are involved in the endogenous metabolism of leukotrienes and prostaglandins [START_REF] Jakobsson | Identification of human prostaglandin E synthase: A microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target[END_REF].

Figure 1 :

 1 Figure1: General scheme of the glutathione transferase (GST) catalysed detoxification of organic xenobiotics and endobiotic metabolites and its impairment by metals at toxic concentration, either by direct inhibition of enzyme activity* or indirectly by decreasing the concentration of the GST substrate glutathione (GSH) via changing the GSH/GSSG equilibrium**.

Figure 2 :

 2 Figure 2: A graphical overview of the different classes of glutathione transferase (modified from Kumar and Trivedi [16], Copyright © 2018 Kumar and Trivedi, doi: 10.3389/fpls.2018.00751).

Figure 3 :

 3 Figure3: Measurement of the GST activity and its inhibition by metals by using the model xenobiotic CDNB (1-chloro-2,4-dinitrobenzene). GST catalyses the reaction of reduced GSH with CDNB via a nucleophilic aromatic substitution, the absorbance of the resulting GS-conjugate measured at 340 nm indicates the GST activity.

Figures 370 Figure 1

 3701 Figures

Figure 2 375Figure 3

 23 Figure 2

  385 a PMS of kidney homogenate EPNP = 1,2-epox-y-3-(p-nitrophenoxy)-propane DCNB = 1,2-dichloro-4-nitrobenzene CDNB = 1-chloro-2,4-dinitrobenzene EA = ethacrynic acid 390

Table 1 .

 1 In vitro and in vivo inhibitory effects of metals on cytosolic GST* activity 380

	Metal or	Exposure Acceptor	Enzyme source	Inhibition IC50	Ref.
	metal		substrate		(μM)
	compound
	Animals
	Al 3+	100 mg/kg CDNB	male Sprague Dawley rats	68%	[43]
	Al 3+	172.5 mg/kg CDNB	female Wistar rats	24%	[44]
	Cr 6+	10 mg/L CDNB	Frogs (Rana ridibunda)	58%	[50]
	Cu 2+		CDNB	Rat liver cytosol	0.23	[55]
					in the presence of
					1 mM ascorbate
	Cu 2+	100 µM CDNB	Mullet (Liza saliens) liver cytosol	61%	[82]
	Cu 2+		EA		51%	[82]
	Cu 2+	100-800	CDNB	Protaetia brevitarsis Lewis	37%-45%	[54]
		mg/kg		larvae
	As 3+ (NaAsO2)	10 mg/kg CDNB	Swiss mice	53%	[56]
	As 3+ (As2O3)	500 μg/L CDNB	Worm (Laeonereis acuta)	48%	[59]
	Sb 3+	100 µM CDNB	Mullet (Liza saliens) liver cytosol	69%	[82]
	Sb 3+		EA		69%	[82]
	SeO3 2-	100 μg/L CDNB	Tilapia fish (Oreochromis	20%	[62]
				mossambicus)
	Ag +	15 µM CDNB	Van Lake fish (Chalcalburnus	11	[65]
				tarichii)
	Ag +	60 µM CDNB	Erythrocytes (rainbow trout,	35	[64]
				Oncorhynchus mykiss)
	Ag +	2 µM CDNB	Turkey liver	0.4	[67]
	Cd 2+	100 µM CDNB	Mullet (Liza saliens) liver cytosol	65%	[82]
	Cd 2+		EA		78%	[82]
	Cd 2+	100 µM CDNB	Rabbit (Oryctolagus cuniculus)	53%	[85]
				lung cytosol
	Cd 2+	2.5 mg/kg CDNB	Balb/C mice	35%	[74]
	Cd 2+	1 mg/kg CDNB	Plaice (Pleuronectes platessa)	90%	[102]
	Cd 2+	800 µM CDNB	Murine hepatocytes	41%	[76]
	Cd 2+	116 mg/L CDNB	Freshwater crab (S.	44%	[103]
				yangtsekiense)
	Tributyltin		CDNB	Rabbitfish a (Siganus	0.92	[78]
				canaliculatus)
	Tributyltin		CDNB	Seabream a (Sparus sarba)	3.5	[78]
	Tributyltin	0.3 mg/kg CDNB	Arctic charr (Salvelinus alpinus)	70%	[80]
	Dibutyltin		CDNB	Rabbitfish a (Siganus	3.2	[78]
				canaliculatus)
	Triphenyltin		CDNB	Rabbitfish a (Siganus	4	[78]
				canaliculatus)
	Triphenyltin			Seabream a (Sparus sarba)	1.8	[78]
	Hg 2+	100 µM CDNB	Mullet (Liza saliens) liver cytosol	70%	[82]
	Hg 2+		EA		86%	[82]

Acknowledgements

This work was supported by the bilateral (German-French) DAAD -Campus France program PROCOPE funded by the German 'Bundesministerium für Bildung und Forschung (BMBF)', the French 'Ministère des Affaires étrangères et du développement International (MAEDI)' and 'Ministère de l'Education nationale, de l'Enseignement supérieur et de la Recherche (MENESR)'.

Conflict of interest

The authors declare no conflict of interests.


1 Secondary Ion Mass Spectrometry for Biological Applications # Dirk Schaumlöffel CNRS / Université de Pau et des Pays de l'Adour / E2S UPPA, Institut des Sciences Analytiques et de Physico-Chimie pour l'Environnement et les Matériaux, UMR 5254, 64000 Pau, France ; dirk.schaumloeffel@univpau.fr With this brief editorial, I want to introduce the themed collection on Secondary Ion Mass Spectrometry (SIMS) for Biological Applications. The selected articles of this collection are highlighting one major feature of SIMS applied to biology: the ability to acquire high-resolution element and molecule specific images of cells and tissues. Many readers of JAAS are probably more familiar with the technique of Laser Ablation (LA) ICP-MS for generation of elemental images from biological tissues; therefore, this themed collection aims to introduce SIMS, which is an atomic spectrometry technique, too, as important tool for bioimaging.

Imaging and imaging techniques are a steadily growing field of high interest in science. This great success is surely conditioned by the fact that we as humans are much more perceptive when information is visualized. For example, this is expressed by the well-known saying that "a picture is worth a thousand words". Indeed, images allow us a quick absorption of large amounts of data. Already in the dawn of humankind, men used images such as cave paintings as an effective way to communicate abstract and concrete information. In science, visual observation has always been an important way to acquire new knowledge followed by its transmission with the help of images. Therefore, the development of light microscopes in the 16 th and 17 th century was a major milestone in order to get insights into the microworld. From the beginning, researchers used these new tools for observation of biological organisms. This first bioimaging led to the early description of biological cells, recorded by Robert Hook's drawings in Microscopia (1665). Nowadays, powerful electron microscopes, notably transmission electron microscopes (TEM), allow the imaging of subcellular structures and small objects such as viruses. Highresolution TEM with correction of spherical aberrations can achieve a resolution down to 0.08 nm enabling even the visualization of atoms.

Although light and electron microscopy give already fascinating insights into small biological structures on micro and nanometer level, there is a considerable interest to combine this structural information with chemical information. The question is How to obtain information on the presence and the quantity of chemical elements including their isotopes as well as molecules in each pixel of an image? For example, TEM can be combined with energy-dispersive X-ray spectroscopy (TEM/X-EDS) preserving almost the high resolution of TEM alone but with element sensitivity only in the percentage range and no possibility of isotope analysis. On the other side, LA ICP-MS provides high element sensitivity at low µg/kg level but only low spatial resolution at the micrometer level, which is not sufficient for subcellular imaging. The gap between these two opposite possibilities is filled by the SIMS techniques. In SIMS imaging a primary ion beam is scanning the sample surface and generating secondary ions of the chemical elements or molecules which are subsequently analyzed by a mass spectrometer. SIMS provides high spatial resolution down to 50-100 nm sufficient for the observation of subcellular structures combined with element sensitivity at the mg/kg level as well as the possibility of isotope analysis.

The present themed collection provides to the reader three review articles and six research papers from different application fields of SIMS bioimaging. Notably, the tutorial and critical reviews of this collection are presenting and comparing different SIMS techniques. A first tutorial review introduces the two main different SIMS configurations time-of-flight SIMS (ToF-SIMS) and nanoscale SIMS (NanoSIMS) as well as some new developments, correlative imaging with other imaging techniques, and applications in neurobiology and cell biology. Briefly, ToF-SIMS uses a pulsed primary ion beam, which ionized the first atomic layers of the sample surface. It allows the detection of element and molecule ions separated by a time-of-flight mass analyzer. In contrast, NanoSIMS employs a continuous high energy and highly focused ion beam that ionizes several nanometers of the sample surface but produces only atomic ions, which are separated with a magnetic sector mass analyzer. A second tutorial review focuses more in detail on ToF-SIMS regarding different primary ion beams, signal enhancement methods, identification of biomolecules (lipids, peptides, proteins, and DNA), single cell imaging, and the possibilities to generate threedimensional images. The critical review explores the applications of NanoSIMS in biology and biomedical research. This includes the detection of non-metals such as N, P, S, and Br in cells and tissues. A special application of NanoSIMS is the use of stable isotopes such as 2 H, 13 C, 15 N, and 18 O for tracking of intracellular metabolic processes. Examples are the investigation of nutrient fluxes, nucleic acid synthesis, lipid and glucose metabolism, and protein turnover.

Three research papers focus on ToF-SIMS applications. One paper deals with the detection of zinc the hippocampus of rat brain after traumatic brain injury. Two papers are exploring the possibilities of threedimensional ToF-SIMS imaging; one demonstrates the localization of titanium dioxide nanoparticles in algal biofilm with high lateral resolution of about 100 nm, the other paper presents a study on lipid composition and their 3D distribution in the ovaries of mosquitos.

Although the number of NanoSIMS instruments worldwide that are dedicated to biological applications is relatively limited, two research papers with NanoSIMS applications could be included in this collection. One paper presents the development of fluorinated nanobodies for specific labeling of cellular proteins. Via 19 F detection in NanoSIMS the distribution of various target proteins could be visualized in cells. The other paper shows the combination of subcellular arsenic imaging in seaweed with arsenic speciation analysis by coupling techniques. Surprisingly, most arsenic was located in the cell walls and no arsenic was detected inside the cells. In a further paper an IMS-3f SIMS instrument was used to investigate intracellular calcium stores in pig kidney cells. In addition, the use of the stable 44 Ca isotope allowed the imaging of calcium influx in cells.

In summary, this themed collection encompasses the presentation of different SIMS techniques for bioimaging and shows a variety of different biological applications. All papers demonstrate that SIMS bioimaging is not a stand-alone technique but in most cases combined with other imaging tools such as light, fluorescence, and electron microscopy. This reveals the need for correlative imaging and sophisticated data treatment. Other common analytical challenges in SIMS bioimaging concern the development of dedicated sample preparation methods and precise quantification methods. The latter is still difficult due to matrix effects and a lack of matrix-matched standards. The application of chemical imaging by SIMS techniques to biological questions is a rapidly growing field. In future, many more research papers with applications to medicine, pharmacology, toxicology, and environmental sciences are expected.

Finally, I wish to thank all authors who contributed to this themed collection, also the reviewers for their time to evaluate the manuscripts, and the JAAS editorial staff for their work on this collection. I hope the readers will enjoy the articles and that they find many new ideas and stimulation for their own research.


Andrea A Piarristeguy
email: andrea.piarristeguy@umontpellier.fr

Rozenn Le Parc

Michel Ramonda

Raphaël Escalier

Annie Pradel

Topological Study of Phase-Separated Ag-Conducting Chalcogenide Glasses Using Peak Force Quantitative Nano-Mechanical Characterization Q1 Q3

Keywords: Ag-doped chalcogenide glasses, phase separation, Ionic conduction, near field microscopy, nano-mechanical mapping

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Q15

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Q17

Please provide the city name for " [START_REF] Kozicki | Programmable metallization cell memory based on Ag-Ge-S and Q17 Cu-Ge-S solid electrolytes[END_REF]."

Q18

Please provide doi for the following references.

" [START_REF] Cervinka | A contribution to the structure of Ge-Se-Ag glasses[END_REF][START_REF] Balan | Phase separation and ionic conductivity: an electric force microscopy investigation of Q18 silver chalcogenide glasses[END_REF]." Peak Force Quantitative Nano-Mechanical property mapping (PF-QNM) was used to explore the nanomechanical properties of binary Ge z Se 100-z glasses and phase-separated Ag x (Ge 0.25 Se 0.75 ) 100-x glasses. The indentation modulus decreases when the selenium content increases in the binary glasses and when the silver content increases in phase-separated ternary glasses as a consequence of the increasing flexibility of the network. At nanoscale level, PF-QNM measurements highlighted a higher indentation modulus for the Ag-rich phase than for the Ag-poor phase, together with a decrease of both local indentation moduli with the increase of silver content in the glass.

A model to gain a further insight in the mean coordination numbers and compositions of the Ag-rich and Ag-poor phases, based upon the assumption of a decoupled effect of the variations in Ag and in Ge on the mechanical properties, is described in detail. In the framework of the model, the mean coordination numbers for the Ag-rich phase <N R > and Ag-poor phase <N P > keep on decreasing when the total silver content in the glasses increases. The Ge concentration differs in Ag-rich and Ag-poor phases. When the silver content in the glass increases, the Ge concentration decreases in both phases. The result, in turn, indicates that an increase in the conductivity of the Ag-rich phase when the total silver content increases, as shown previously by C-AFM, would not be only explained by an increase in silver content but also by a softening of the network and consequently by an increase of charge carrier mobility.

INTRODUCTION

Owing to their remarkable properties (IR transparency, high ionic conduction, photo-diffusion, Q13 Q6 Q8 etc), chalcogenide glasses aroused strong interest in both the academic community and private companies. Among potential applications, the Conductive-Bridging Random Access Memories (CB-RAM), susceptible to replace the current flash memories, take advantage of two properties their high ionic conductivity when doped with silver. A CB-RAM memory typically comprises a Ag-photo-doped glassy thin film of composition ∼Ge 0.25 Se 0.75 placed between two electrodes, a silver one and a nickel one for example. The conductivity of the film is reversibly changed by several orders of magnitude when a low voltage is applied (∼0.2V). When applied to nanometric devices (when the thickness of the glassy film is typically 20-30 nm), the phenomenon is characterized by a very short time for commutation (∼10 ns) and a very high cyclability (>10 6 cycles) [START_REF] Kozicki | Programmable metallization cell memory based on Ag-Ge-S and Q17 Cu-Ge-S solid electrolytes[END_REF].

In order to explain the operation of this memory and in particular its very short switching time hardly compatible with the mobility of Ag + ions, various hypotheses have been proposed and among them, the presence of pre-existing silver-rich zones within the amorphous film-which would be heterogeneousand the creation of conduction paths between these zones. To confirm this hypothesis and to better understand the phenomena at the origin of the switching from a resistive state to a conductive state in CB-RAM memories, the electrical, structural, and thermal properties of bulk Ag-Ge-Se glasses have been widely studied [START_REF] Dejus | The structure of silver/germanium/selenium glass[END_REF][START_REF] Dejus | Dynamics of vitreous Ag-Ge-Se[END_REF][START_REF] Dejus | Structure of vitreous AgGeSe[END_REF][START_REF] Kawasaki | Ionic conductivity of Ag x (GeSe 3 ) 1-x (0 ≤ x ≤ 0.571) glasses[END_REF][START_REF] Mitkova | Dual chemical role of Ag as an additive in chalcogenide glasses[END_REF][START_REF] Iyetomi | Incipient phase separation in Ag/Ge/Se glasses: clustering of Ag atoms[END_REF][START_REF] Piarristeguy | X-ray analysis of GeSeAg glasses[END_REF][START_REF] Piarristeguy | Structural considerations about the (Ge 0.25 Se 0.75 ) 100-x Ag x glasses[END_REF]Piarristeguy et al., , 2007a[START_REF] Piarristeguy | Short range order and Ag diffusion threshold in Ag x (Ge 0.25 Se 0.75 ) 100-x glasses[END_REF][START_REF] Ureña | Crystallization processes of Ag-Ge-Se superionic glasses[END_REF][START_REF] Ureña | Ionic conductivity (Ag + ) in AgGeSe glasses[END_REF][START_REF] Wang | Macroscopic phase separation of Se-rich (x < 1/3) ternary Ag y (Ge x Se 1-x ) 1-y glasses[END_REF][START_REF] Cervinka | A contribution to the structure of Ge-Se-Ag glasses[END_REF][START_REF] Tafen | Silver transport in Ge x Se 1-x :Ag materials: Ab initio simulation of a solid electrolyte[END_REF][START_REF] Cuello | Structure of chalcogenide glasses by neutron diffraction[END_REF][START_REF] Prasai | Ab initio simulation of solid electrolyte materials in liquid and glassy phases[END_REF][START_REF] Parc | Ag-Ge-Se glasses: a vibrational spectroscopy study[END_REF]Stellhorn et al., 2016a,b;[START_REF] Zeidler | Structure of glassy Ag-Ge-Se by neutron diffraction with isotope substitution[END_REF][START_REF] Zeidler | Structure of semiconducting versus fast-ion conducting glasses in the Ag-Ge-Se system[END_REF]. It was shown that the conductivity of Ag x (Ge 0.25 Se 0.75 ) 100-x glasses with 1 < x < 30 at. % increases suddenly by seven to eight orders of magnitude at 7-10 at. % Ag.

Insight into the microstructure of these glasses was clearly needed. However, owing to the ease of diffusion of silver in these materials [START_REF] Kolobov | Photodoping of amorphous chalcogenides by metals[END_REF], the use of classical microscopy techniques to probe the structure of Ag-based glasses is tricky; a risk of altering the Ag-centered local structures exists if the light or electron beam energy is too high. As a matter of fact, few papers on the electrical properties of chalcogenide glasses report attempts to get insights into the homogeneity of the materials. [START_REF] Gutenev | Dielectric loss in Ag-Ge-Se system glasses[END_REF] and [START_REF] Wang | Macroscopic phase separation of Se-rich (x < 1/3) ternary Ag y (Ge x Se 1-x ) 1-y glasses[END_REF] indicated for the first time that the glasses were phase separated according to optical microscopy and modulated differential scanning calorimetry (MDSC) experiments, respectively. However, these experiments cannot provide insight on, for example, the evolution of the composition or the conductivity of each phase with increasing silver content. Several years later, the ability and the complementarity of near field microscopy techniques provided insight into the intrinsic electrical properties of each phase present in bulk Ag x (Ge 0.25 Se 0.75 ) 100-x glasses and their composition dependence [START_REF] Balan | Phase separation and ionic conductivity: an electric force microscopy investigation of Q18 silver chalcogenide glasses[END_REF][START_REF] Piarristeguy | Phase separation in Ag-Ge-Se glasses[END_REF]Piarristeguy et al., , 2010a,b),b). Electrostatic force microscopy (EFM) was indeed used to get images of electrical contrasts (dielectric permittivity) in phase separated glasses. Combined with electrical conductivity measurements using complex impedance spectroscopy, EFM helped in understanding the jump in conductivity at 7-10 at% in Ag x (Ge 0.25 Se 0.75 ) 100-x glasses. While a Ag-rich phase is embedded in a Ag-poor phase for the low conductivity region (x < 7-10 at. % Ag), the reverse is true for high conductivity region (x > 7-10 at. % Ag) with the Ag-rich phase controlling the silver diffusion throughout the material. The percolation of the Ag-rich phase was at the origin of a sudden jump in conductivity [START_REF] Balan | Phase separation and ionic conductivity: an electric force microscopy investigation of Q18 silver chalcogenide glasses[END_REF][START_REF] Piarristeguy | Phase separation in Ag-Ge-Se glasses[END_REF].

Recently another near field microscopy technique, i.e., the Contact Resonance Atomic Force Microscopy (CR-AFM) in combination with Raman mapping was used to get additional information on the phase compositions in bulk Ag x (Ge 0.25 Se 0.75 ) 100-x glasses [START_REF] Piarristeguy | Local vibrational and mechanical characterization of Ag conducting chalcogenide glasses[END_REF]. The results suggested a different Ge/Se ratio in Ag-poor and Ag-rich phases, the first one being richer in selenium than the second one.

In this work, an innovative and complementary technique, the Peak Force Quantitative Nano-Mechanical mapping (PF-QNM) was used to explore the nano-mechanical properties of Ge y Se 100-y and Ag x (Ge 0.25 Se 0.75 ) 100-x glasses and get information on the topology of these phase-separated glasses. A model, based upon the assumption of a decoupled effect of the variations in Ag and in Ge on the mechanical properties, helped to gain a further insight in the mean coordination numbers and compositions of the Ag-rich and Ag-poor phases.

MATERIALS AND METHODS

Sample Preparation and Characterization

Bulk Ge y Se 100-y glasses (y = 15, 20, 25, 30, and 33 at. %) and Ag x (Ge 0.25 Se 0.75 ) 100-x glasses (x = 0, 7, 15, and 20 at. %) were synthesized from a mixture of high-purity starting elements [Ag (Aldrich, 99.9 %), Ge (Aldrich, 99.999 %) and Se (Aldrich, 99.99 %)]) Three grams of the stoichiometric powders were placed in a cylindrical quartz ampoule (11 mm inner diameter, 1 mm thick) sealed under secondary vacuum (10 -5 mbar). The tube was subsequently heated with a low heating rate of 6 • C/h up to 600 • C for Ge 15 Se 85 , 700 • C for Ge 20 Se 80 and 850 • C for Ge 30 Se 70 and Ge 33 Se 67 , held at this temperature for 12 h, and finally quenched in air. For the Ag x (Ge 0.25 Se 0.75 ) 100-x glasses, the powder was heated up to 950 • C with a 10 • C/h heating rate and kept at this temperature for 8 h for homogenization before being quenched in a salt-ice-water mixture. The samples were named Agx according to the concentration of Ag (in at. %) and the nomenclature R-Agx and P-Agx was used for the Ag-rich and Ag-poor phases, respectively.

The amorphous nature of the ingots was confirmed by performing X-Ray diffraction using a PANalytical X'Pert diffractometer. A Cu (Kα) source (λ = 1.5406Å) was used with an operating voltage of 40 kV and a beam current of 30-40 mA.

Peak Force Quantitative Nano-Mechanical Property Mapping (PF-QNM)

The mechanical characterization was performed with a Multimode AFM instrument (from Bruker Corporation, USA) upgraded with Nanoscope V using PF-QNM imaging mode and a special RTESPA-525-30 (BatchA050 wafer1 Box9 probe#3) (Bruker) probe with a lever spring constant of 167 N/m and a tip radius of 34 nm. A FSILICA-12M sample from Bruker Instruments was first used to calibrate sensitivity deflection and sync distance QNM. The calibration was checked by measuring a sample (given by [START_REF] Arnould | Better insight into the nano-mechanical properties of flax fibre cell walls[END_REF] consisting in a mixture of resin and fiber of K28 grade Kevlar, providing indentation moduli of around 4 GPa ± 0.4 GPa and 23 GPa ± 4 GPa, respectively, in agreement with the data of [START_REF] Arnould | Better insight into the nano-mechanical properties of flax fibre cell walls[END_REF]. The applied maximum load was set at 200 nN for all the measurements. Different mechanical properties, i.e., adhesion force, elastic modulus, deformation, energy dissipation, were obtained by a real time analysis of the "force versus separation" curves. The well-known Derjaguin-Muller-Toporov (DMT) model [START_REF] Derjaguin | Effect of contact deformations on the adhesion of particles[END_REF] was used to fit the retract curves and calculate the indentation moduli (Poisson coefficient set at 0).

Measurements were performed on the surfaces of freshly fractured glass to avoid any problem due to surface oxidation. The experiments were carried out within the following hours.

RESULTS

Ge-Se Glasses

PF-QNM experiments allowed getting information about the mechanical properties of glasses. The indentation moduli, reported for glasses for the first time, were estimated from data recorded over 5 × 5 µm images. Figure 1 shows the indentation moduli for the different Ge y Se 100-y glasses. A monotonous increase in modulus with increasing germanium concentration (y) is observed, which is in agreement with Vickers and Meyer's hardness and Young's modulus measured by conventional mechanical techniques [START_REF] Guin | Hardness, toughness, and scratchability of germanium-selenium chalcogenide glasses[END_REF]. The insert in Figure 1 shows both the indentation moduli obtained by PF-QNM measurements and the Young's moduli for the same glassy compositions [START_REF] Guin | Hardness, toughness, and scratchability of germanium-selenium chalcogenide glasses[END_REF]. Similar trends are observed, which allows to validate the PF-QNM technique to evaluate realistic glass indentation moduli.

Because of similar Se-Se and Se-Ge bond strengths (223 and 230 kJ/mol, respectively) and only slightly weaker Ge-Ge bond strength (186 kJ/mol), [START_REF] Guin | Hardness, toughness, and scratchability of germanium-selenium chalcogenide glasses[END_REF] claimed that the source Q14 for the increase of the hardness has to be topological in nature. Consequently, the continuous increase of hardness is of the same origin as the increase of the elastic moduli, and it results from the increasing number of strong covalent bonds with increasing Ge concentration.

Ag-Ge-Se Glasses

Figure 2 shows the topography and the indentation modulus images (PF-QNM) for the Ag x (Ge 0.25 Se 0.75 ) 100-x glasses. These images (Figures 2b,d,f) clearly show heterogeneous domains, which indicates that the PF-QNM technique is sensitive to modulation of mechanical properties at the local stage. The heterogeneous domains are similar to those already reported for this family of glasses using Electrostatic Force Microscopy (EFM) and Conductive-Atomic Force Microscopy (C-AFM) [START_REF] Balan | Phase separation and ionic conductivity: an electric force microscopy investigation of Q18 silver chalcogenide glasses[END_REF][START_REF] Piarristeguy | Phase separation in Ag-Ge-Se glasses[END_REF]Piarristeguy et al., , 2010a,b),b). For glassy compositions belonging to the low-conductivity region (x < 10 at. % Ag, Figure 2b), nodules of a Ag-rich (R-Agx) phase (light region) are embedded in a connected Agpoor (P-Agx) phase (dark region). At the opposite, for the glassy compositions belonging to the high conductivity region (x=15 and 20), a connected R-Agx phase contains nodules of P-Agx phase (Figures 2d,f). Depending upon the quenching conditions, spinodal or nucleation/growth phase separation can occur in these glasses [START_REF] Piarristeguy | Local vibrational and mechanical characterization of Ag conducting chalcogenide glasses[END_REF]. In this work, the images show a morphology typical of nucleation/growth phase separation.

As an example, the analysis of a small zone of the indentation modulus mapping for a typical phase-separated Ag 20 (Ge 0.25 Se 0.75 ) 80 glass is detailed (Figure 3). This glass presents nodules of about 0.7 µm (P-Agx phase) immersed in a conductive matrix (R-Agx phase). The indentation moduli of both phases are visible in Figure 3A. The variation of modulus along the white line in Figure 3A is shown in Figure 3B. The modulus of the R-Agx phase, i.e., ∼9 GPa, corresponds to the domains of the line from 0 to ∼0.5 µm and from ∼1.3 to 2 µm. The modulus for the R-Agx phase between ∼0.5 and ∼1.3 µm has a value of ∼7.2 GPa.

Three different values of indentation modulus for each of these phase-separated glasses were extracted from the PF-QNM experiments. The first one corresponds to an average value and is evaluated by taking into account the whole region included in the blue parallelepiped in Figure 4, the second one corresponds to the indentation modulus for the R-Agx phase and is obtained from regions included in the red parallelepiped in Figure 4, the third one corresponds to the P-Agx phase (regions in the green parallelepiped in Figure 4). The average indentation modulus, estimated from data recorded over the whole 5 × 1.5 µm image is equivalent to a "macroscopic measurement." At least thirty different regions were taken into in the PF-QNM image to estimate the value of and the uncertainty on the indentation modulus for each phase. The indentation moduli for R-Agx and P-Agx phases as well as the "average modulus" as a function of the silver content are presented in Figure 5. The "average modulus" decreases with the increase of silver concentration in the glass, which is in agreement with previous Vickers microhardness measurements [START_REF] Piarristeguy | Local vibrational and mechanical characterization of Ag conducting chalcogenide glasses[END_REF]. This decrease is a consequence of the structural changes induced by the introduction of Ag in the Ge 25 Se 75 glass. Introduction of silver affects the tetrahedral network through changes in bond angles, eventual ES to CS tetrahedra transformation associated to network disruption and selenium chains breaking. At high silver content, a high flexibility of the network is expected as a consequence of depolymerization of the glass network. Based on this statement, the local mechanical measurements present an apparent contradiction since the indentation modulus value is higher for the R-Agx phase than for the P-Agx phase (it will be discussed in the next section). A decrease of both local indentation moduli with the increase of the silver content in the glass is also observed.

DISCUSSION

Mean Coordination Number and Composition

The results above show a decrease in the "average" indentation modulus M (symbols in blue, Figure 5) when the Ag content increases in the glasses. However, for a given Ag content, the FIGURE 5 | Indentation moduli for bulk Ag x (Ge 0.25 Se 0.75 ) 100-x glasses. Blue, red and green symbols correspond to the "average modulus," R-Agx and P-Agx phases moduli, respectively. The broken lines are drawn as guides for the eye. The indentation modulus values and the error bars were calculated from: i) an average of three PF-QNM measurements and standard deviation evaluation for the "average modulus" and ii) an average of PF-QNM measurements over thirty different regions (see green and red regions in Figure 4) and subsequent standard deviation evaluation for the Ag-rich and Ag-poor phases.

R-Agx phase systematically exhibit a higher modulus than the P-Agx phase. The explanation has to be searched in difference in compositions and, consequently in average coordination numbers, between R-Agx and P-Agx.

In Ge y Se 100-y glasses, assuming <N Ge > = 4 and <N Se > = 2 for the mean coordination numbers of Ge and Se, respectively, the mean coordination number can be estimated

< N > Ge-Se = y 100 < N Ge > + 100 -y 100 < N Se > (1)
In Ag x (Ge 0.25 Se 0.75 ) 100-x glasses, when ionic bonds are participating to the structure, the determination of the mean coordination is more complex. Zeidler et al. ( 2018) have estimated the nearest-neighbor coordination numbers <N ij > extracted from neutron diffraction experiments on glasses along the Ag x (Ge 0.25 Se 0.75 ) 100-x tie line. Taking into account that the first coordination sphere implies only <N GeSe >, <N SeSe >, and <N SeGe >, the mean coordination number for this family of glasses can be estimated

< N > Ag-Ge-Se = 0.25(100 -x) 100 < N GeSe > + 0.75(100 -x) 100 [< N SeGe > +< N SeSe >] (2)
Figure 6 shows the mean coordination number <N> for Ge y Se 100-y and Ag x (Ge 0.25 Se 0.75 ) 100-x glasses. As expected, we observe that the mean coordination number decreases monotonously with the increase of silver content in the glass, with values comprised between <N> = 2.50 for Ag0 to <N> = 1.62 for Ag25. Silver incorporation is expected to depolymerize the network, searching for selenium to compensate its positive charge, leading to inter-tetrahedral Ge-Se-Ge, and selenium chains breaking. Therefore, the introduction of silver is expected to introduce some flexibility into the network.

An attempt to gain a further insight into the mean coordination numbers of the R-Agx and P-Agx phases in Ag x (Ge z Se 1-z ) 100-x glasses is described below.

As a first approximation, the dependence of the indentation modulus M Ge on Ge content in Ge y Se 100-y glasses, shown in Figure 7 (black symbols), can be taken as linear with M Ge = 14.04 < N >

(3)

In the same way, the dependence of the indentation modulus M Ag on Ag content for Ag x (Ge 0.25 Se 0.75 ) 100-x glasses, shown in Figure 7 (blue symbols), can be taken as linear with

M Ag = -3.94 < N > (4)
On the whole, the effect of Ge modulation is almost three times more impacting on the indentation modulus than the evolution of the average Ag content and evolves in the opposite direction.

With the assumption that the difference of indentation moduli M R-P = M(R-Agx)-M(P-Agx) between the Agrich phase (Ag x ′ (Ge z ′ Se 1-z ′ ) 100-x ′ ) and the Ag-poor phase (Ag x ′′ (Ge z ′′ Se 1-z ′′ ) 100-x ′′ ), for a given glass composition, can be related to the decoupled effect of both the variation in Ag content,

x ′ x ′′ = x ′ -x ′′ , and the variation in Ge content, z ′ z ′′ = 100.z ′ -100. z ′′ , one can write where M x ′ x ′′ is the modulus difference related to a Ag variation x ′ x ′′ and M z ′ z ′′ a modulus difference related to Ge variation z ′ z ′′ . Taking into account Equations (3-5) can be written M R-P = 14.04 < N > -3.94 < N >= 10.06 < N > (6) The modulus difference between R-Agx and P-Agx phases, M R-P , for a given glass, can be calculated from the experimental data shown in Figure 5. These values are 2.33, 1.85, 1.43, for Ag7, Ag15, and Ag20 glasses respectively, which corresponds (see Equation 6) to <N> values of 0.23, 0.18, and 0.14 with the Ag content increasing in the glass.

M R-P = M x ′ x ′′ + M z ′ z ′′ (
Additional information can help to estimate the mean coordination number for R-Agx (<N R >) and P-Agx (<N P >) phases. The PF-QNM images (Figures 2b,d,f) indeed allow to calculate the surface percentage for the R-Agx (% R) and P-Agx (% P) phases. Assuming that the volume percentage is equivalent to the surface percentage, the mean coordination number is therefore a linear combination of the mean coordination number in R-Agx and P-Agx phases:

< N >= (%P) < N P > + (%R) < N R > = (%P) < N P > + (%R)(< N P > + < N >) (7)
where we have assumed that <N R > = <N P > + <N> because R-Agx phase presents a high indentation modulus in comparison with the P-Agx phase. The mean coordination numbers <N R > and <N P > are presented in Figure 7.

It can be seen that, consistently with the average coordination number, both <N R > and <N P > decrease with increasing Ag content. These decreases in coordination number can be understood by a decrease in Ge (z ′ , z ′′ ) and/or an increase of Ag (x ′ , x ′′ ). The differences <N R > -<N P > for a specific Agx composition decreases when Ag content increases (respectively equal to 0.23, 0.18, 0.14 for Ag7, Ag15, and Ag20). Interestingly, depending upon the preparation procedure, the glass Ag 25, not measured here, is either homogeneous or shows only very small inhomogeneities.

As a matter of fact, compositions of the P-Agx and R-Agx phases should not only differ in Ag composition (x ′ , x ′′ ), but also in Ge composition (z ′ , z ′′ ) as already suggested by the results from CR-AFM data shown in a previous [START_REF] Piarristeguy | Local vibrational and mechanical characterization of Ag conducting chalcogenide glasses[END_REF]. Indeed, one expects a R-Agx phase with a higher Ge content (z ′ ) than the P-Agx one (see Figure 8).

In the following, we aim at gaining a further insight in the compositions of the R-Agx phase (Ag x ′ (Ge z ′ Se 1-z ′ ) 100-x ′ ) and of the P-Agx phase (Ag x ′′ (Ge z ′′ Se 1-z ′′ ) 100-x ′′ ). As already mentioned, according PF-QNM images (Figures 2b,d,f), the average composition is a linear combination of the compositions in R-Agx and P-Agx phases:

Ag x (Ge 0.25 Se 0.75 ) 100-x = (%R) Ag x ′ Ge z ′ Se 1-z ′ 100-x ′ + (%P) Ag x ′′ Ge z ′′ Se 1-z ′′ 100-x ′′ (8) with x = (%R) x ′ + (%R) x ′ ′ (9) 0.25 (100 -x) = (%R) z ′ 100 -x ′ + (%P) z ′ ′ 100 -x ′ ′ (10)
where (% R) and (% P) correspond, respectively to the surface percentage for R-Agx and P-Agx phases.

Combining these equations leads to a great number of potential compositions, however further restrictions on the FIGURE 8 | Indentation moduli for R-Agx (red symbols) and P-Agx (green symbols) phases in Ag x (Ge 0.25 Se 0.75 ) 100-x glasses and for Ge y Se 100-y glasses (black symbols). Percentages correspond to the proportion of R-Agx and P-Agx phases for each composition Agx. The broken lines are drawn as guides for the eye.

Ge content can be considered and reduce the number of suitable compositions.

At first, the Ge content for the R-Agx phase (z ′ ) has to be larger than 0.25 since it was shown that the R-Agx phase is richer in Ge than the P-Agx one, and obviously to the average composition (z). A second set of restrictions can be estimated from the difference of indentation moduli M R-P = M(R-Agx)-M(P-Agx) between R-Agx and P-Agx phases, assuming once again that the modulus dependence on Ag and Ge can be decoupled (Equation 5).

M R-P = -0.12 x ′ x ′′ + 0.28 z ′ z ′′ (11)

where M Ge = 0.28 y = 0.28 z ′ z ′′ and M Ag = -0.12 x = -0.12 x ′ x ′′ were obtained from a linear dependences of the indentation modulus M Ge on Ge content in Ge y Se 100-y glasses (Figure 1) and the indentation modulus M Ag on Ag content for Ag x (Ge 0.25 Se 0.75 ) 100-x glasses, shown in Figure 5 (blue symbols).

With the above information, it is possible to access to the possible values z ′ z ′′ for each Agx composition. Figure 9 shows the indentation modulus differences M R-P predicted as a function of x ′ x ′′ in the form of several linear curves, each one corresponding to possible values of z ′ z ′′ . Intercepts between experimental M R-P and the line network give access to the potential z ′ z ′′ values for each Agx composition. On can deduce that -For Ag 7: Taking into account all the previous constraints that include z ′ > 0.25, x ′ > x, 0 < x ′′ < x, Equations (8-11) and z ′ z ′′ estimated from Figure 9, several compositions for the R-Agx phase (x ′ and z ′ ) and for the P-Agx phase (x ′′ and z ′′ ) can be proposed. They are reported in Figure 10. Among these compositions, some might FIGURE 9 | Indentation modulus differences M R-P predicted as a function of x ′ x ′′ in the form of several linear curves each one corresponding to possible values of z ′ z ′′ . Horizontal lines correspond to experimental M R-P for Ag7, Ag15, and Ag20.

not be realistic, in particular if they do not contain a sufficient Se ratio to compensate the positive charges brought by Ge and Ag (compositions not shown in Figure 10).

Therefore, a last restriction is proposed inspired from the limit of Ag x (Ge 0.25 Se 0.75 ) 100-x glass forming ability proposed by [START_REF] Zeidler | Structure of semiconducting versus fast-ion conducting glasses in the Ag-Ge-Se system[END_REF]. This limit corresponds to the consumption of all Se-Se homopolar bonds, can be estimated when the concentration of Ge, Se, and Ag lead to the mean coordination number associated to the number of Se-Se bonds n Se Se = 0. Then n Se

Se can be calculated from this relation

n Se Se = Z Se -Z Ge * y 1 -y -2N Broken * 2x (1 -y)(100 -x) (12
) where Z Se and Z Ge are the overall coordination numbers of Se and Ge equal to 2 and 4 respectively, and N Broken is the mean number of broken Se-Se bonds per silver atoms estimated around 0.33 [START_REF] Zeidler | Structure of semiconducting versus fast-ion conducting glasses in the Ag-Ge-Se system[END_REF]. The criteria of n Se Se = 0 has been used to find the limit between the compositions lying within the glass forming ability and those beyond. The composition limit is shown in Figure 10. Most compositions found for R-Agx and P-Agx phases are in the glass forming compositions.

An exact value of Ag concentration in R-Agx and P-Agx phases cannot be given from the previous analyses but some interesting information can be brought: (i) the Ge concentration differs in R-Agx and P-Agx phases with Ge concentration being larger in R-Agx phase, (ii) when the average silver content increases, the Ge concentration decreases in both phases, (iii) z ′ and z ′′ lie in the range 19-31 at. % Ge.

Conductivity

The PF-QNM images (Figure 2) show a nucleation/growth type phase separation in the Ag x (Ge 0.25 Se 0.75 ) 100-x glasses, in agreement with previous papers [START_REF] Balan | Phase separation and ionic conductivity: an electric force microscopy investigation of Q18 silver chalcogenide glasses[END_REF][START_REF] Piarristeguy | Phase separation in Ag-Ge-Se glasses[END_REF]Piarristeguy et al., , 2010aPiarristeguy et al., ,b, 2018)). The heterogeneous structure has been used to explain the sudden jump in conductivity by 8 orders of magnitude at a silver content lying in the range 7-10 at. % Ag. Indeed, while the Ag-rich phase is embedded in a Ag poor phase for the low conductivity region (x < 7-10% at Ag), the reverse is true for the high conductivity region (x > 7-10% at Ag), with the silver rich phase then controlling the silver diffusion throughout the material. Additional nanoscale electrical characterization of the R-Agx phases by conductive atomic force microcopy (C-AFM) showed that the conductivity increased in R-Agx phases with the increase in total Ag content of the glass (x) (Piarristeguy et al., 2010a). Such an increase can be explained by the increase in the Ag content of the R-Agx phase (x ′ ) (see blue, dark green, and red regions in Figure 10). However, the present data also show that with increasing Ag content, a softening of the network in R-Agx phases occurs due to a decrease in Ge (z ′ ). Such a softening could also contribute to an increase in conductivity due to an increase in charge carrier mobility. This proposal agrees with [START_REF] Micoulaut | Fast-ion conduction and flexibility and rigidity of solid electrolyte glasses[END_REF] that states "The ease of conduction in an ion conducting glass could indeed be related to the mechanical nature of the host network."

CONCLUSION

Peak Force Quantitative Nano-Mechanical (PF-QNM) mapping was used to characterize a series of binary Ge z Se 100-z glasses and phase-separated Ag x (Ge 0.25 Se 0.75 ) 100-x glasses. This technique proved to be efficient to obtain mechanical information at the nanoscale in a system where information at the nanoscale are difficult to obtain due to the fast silver photo-diffusion under energetic (light or electron beam) stimulus, leading to modification of Ag-centered local structures. As expected, a decrease in the indentation modulus when the selenium content increased in the binary glasses and when the silver content increased in phase-separated ternary glasses, as a consequence of the increasing flexibility of the network, was observed. At nanoscale level, the sensitivity of PF-QNM measurements allowed to observe a difference in the indentation moduli of the two phases, i.e., high and low for Ag-rich (R-Agx) and Ag-poor (P-Agx) phases, respectively.

A model that contributes to a better understanding of the topology of this phase-separated system has been proposed. It is based on the assumption that the change in nanomechanical properties of the Ag-rich and Ag-poor phases depends on the change in Ag and Ge in an independent way. Within this assumption, the mean coordination numbers and compositions for Ag-rich (R-Agx) and Ag-poor (P-Agx) phases can be qualitatively estimated from PF-QNM data. When the silver content in the glass increases, the mean coordination numbers of the R-Agx phase <N R > and P-Agx phase <N P > decrease continuously. This is accompanied with a decrease of the Ge concentration in both phases but with the average Ge concentration remaining equal to 25.

Finally, these data helped in providing new information about the increase of conductivity in the Ag-rich (R-Agx) phase when silver content increases in the glasses, as observed by C-AFM measurements. As a matter of fact, it would not only be explained by an increase in silver content in the Ag-rich phase but also by a softening of the network and consequently by an increase of charge carrier mobility.

These promising results provide prospects likely to contribute to the understanding of the topology of phase-separated glasses as well as other bi-phase systems such as glass ceramics or composite materials.

  , their sensitivity to photo-diffusion and Q11

FIGURE 1 |

 1 FIGURE 1 | Indentation moduli for bulk Ge y Se 100-y glasses. Insert shows the

FIGURE 2 |

 2 FIGURE 2 | PF-QNM mapping of the Ag x (Ge 0.25 Se 0.75 ) 100-x glasses: (a) x = 7, topography, (b) x = 7, indentation modulus, (c) x = 15, topography, (d) x = 15, indentation modulus, (e) x = 20, topography and (f) x = 20, indentation modulus.

FIGURE 3 |

 3 FIGURE 3 | (A) Detailed mapping of indentation modulus for the Ag 20 (Ge 0.25 Se 0.75 ) 80 glass and (B) variation of the indentation modulus along the profile drawn in (A). In both images, "R" and "P" correspond to R-Agx and P-Agx phases, respectively.

FIGURE 4 |

 4 FIGURE 4 | PF-QNM mapping of indentation modulus for Ag 15 (Ge 0.25 Se 0.75 ) 85 glass. The blue, red and green regions correspond to the average (or macroscopic) indentation modulus, the R-Agx phase and the P-Agx phase modulus, respectively.

FIGURE 6 |

 6 FIGURE 6 | Mean coordination number <N> for Ag x (Ge 0.25 Se 0.75 ) 100-x glasses estimated using Zeidler et al. (2016). For comparison, mean coordination number <N> for Ge y Se 100-y glasses are presented assuming <N Ge > = 4 and <N Se > = 2 for the mean coordination numbers of Ge and Se, respectively.

FIGURE 7 |

 7 FIGURE 7 | Indentation moduli in function of the mean coordination number for Ge y Se 100-y glasses (black symbols), Ag x (Ge 0.25 Se 0.75 ) 100-x glasses (bleu symbols), and R-Agx (red symbols) and P-Agx (green symbols) phases. Percentages correspond to the proportion of R-Agx and P-Agx phase for each composition Agx. The broken lines are drawn as guides for the eye.

  x ′ x ′′ = [0-7], M R-P = 2.33 and z ′ z ′′ = [8.2-11.3] -For Ag 15: x ′ x ′′ = [0-15], M R-P =1.85 and z ′ z ′′ = [6.5-13] -For Ag 20: x ′ x ′′ = [0-20], M R-P = 1.43 and z ′ z ′′ = [5-13.7]

FIGURE 10 |

 10 FIGURE 10 | Curve showing the limiting values of x (Ag) for each composition y (Ge) on the basis of the validity of Criteria of n Se Se = 0. Color regions show compositions found for R-Agx and P-Agx phases.

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ACKNOWLEDGMENTS

The authors acknowledge M. Bigot for its help in sample preparation and O. Arnould and R. Arinero for fruitful

DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/supplementary material. Q15

Conflict of Interest:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.