Thierry Rabilloud
email: thierry.rabilloud@cea.fr

Pierre Lescuyer

Proteomics in mechanistic toxicology: history, concepts, achievements, caveats and potential

Toxicoproteomics can be defined as the application of proteomic approaches to the understanding of toxicology problems, and this review deals with the various types of applications that have been described in the literature. Toxicoproteomics has been applied to very different classes of toxicants, from drugs and natural products to metals, or from industrial chemicals to nanoparticles and nanofibers. It has also been applied to address questions at different levels, from the search of the primary molecular targets of toxicants to the deciphering of the molecular responses of cells and tissues to toxicants. Although restricted to mammalian cells and tissues, this paper reviews these two levels of investigation and the different application areas of toxicoproteomics, leading to the discussion of the advantages and drawbacks of the most popular proteomic platforms. Some of the pending questions in toxicoproteomics are also critically addressed, such as the specificity, validation and result hierarchization issues. The question of shared mechanisms, which are encountered in many toxicoproteomic papers dealing with different toxicants, is also discussed. Finally the future of toxicoproteomics is briefly outlined.

Introduction: history of proteomics in toxicology

One of the major challenges for toxicology is to find how toxicants exert their effects on living organisms. Toxicology needs to provide knowledge of the molecular targets of the toxicants, and also on how an action on a (or a few) target(s) propagate in living cells to give rise to adverse effects. With regard to the complexity of living cells, this appears as a daunting task with almost infinite possibilities of direct and indirect effects. Progressive accumulation of biochemical and physiological knowledge at both the organism and cellular levels has allowed the toxicologists to decipher many mechanisms of toxicity. However, the price to pay in this strategy is that toxicology often follows well-known and well-paved avenues and has difficulties in unraveling new mechanisms or even to put into perspective different putative mechanisms. It is therefore not surprising that with its wide scope and low degree of preconception hypotheses, proteomics would appear as a seducing approach in toxicology. Historically speaking, the first attempts to apply a wide-scope protein screen to toxicology started soon after the first publications of the initial technique of protoproteomics, namely two-dimensional electrophoresis. Those publications were essentially descriptive (e.g. in [START_REF] Johnston | Induction of 4 Proteins in Chick-Embryo Cells by Sodium Arsenite[END_REF][START_REF] Kim | Arsenate Induces Stress Proteins in Cultured Rat Myoblasts[END_REF][START_REF] Anderson | Protein-pattern changes and morphological effects due to methionine starvation or treatment with 5-azacytidine of the phorbolester-sensitive cell lines HL-60, CCL-119, and U-937[END_REF][START_REF] Anderson | Effects of Aroclor-1254 on Proteins of Mouse-Liver -Application of Two-Dimensional Electrophoretic Protein Mapping[END_REF][START_REF] Anderson | Effects of Toxic Agents at the Protein Level -Quantitative Measurement of 213 Mouse-Liver Proteins Following Xenobiotic Treatment[END_REF][START_REF] Witzmann | Determination of Perfluoro-Normal-Decanoic Acid Toxicity Invitro and Invivo Via Two-Dimensional Polyacrylamide-Gel Electrophoresis[END_REF]), because protein identification from 2D gels was an ordeal at that time. Only comigration with a purified know protein, blotting [START_REF] Franke | Isolation and characterization of desmosome-associated tonofilaments from rat intestinal brush border[END_REF], or immunoprecipitation prior to analysis by 2D gels [START_REF] Vlasuk | Polypeptide Patterns of Hepatic Microsomes from Long-Evans Rats Treated with Different Xenobiotics[END_REF], was feasible at that time. Nevertheless, these authors had identified cytochromes P450 [START_REF] Vlasuk | Polypeptide Patterns of Hepatic Microsomes from Long-Evans Rats Treated with Different Xenobiotics[END_REF] and also Hsp proteins [START_REF] Kim | Arsenate Induces Stress Proteins in Cultured Rat Myoblasts[END_REF] as induced by toxicants. With the development of protein identification methods, first by Edman sequencing then by mass spectrometry, it became possible to identify almost every protein spot present on a 2D gels, and modern toxicoproteomics came to birth twenty years ago [START_REF] Witzmann | Induction of Enoyl-Coa Hydratase by Ld(50) Exposure to Perfluorocarboxylic Acids Detected by 2-Dimensional Electrophoresis[END_REF][START_REF] Steiner | Cyclosporine A decreases the protein level of the calcium-binding protein calbindin-D 28kDa in rat kidney[END_REF][START_REF] Aicher | New insights into cyclosporine A nephrotoxicity by proteome analysis[END_REF] (Figure 1). [START_REF] Aicher | New insights into cyclosporine A nephrotoxicity by proteome analysis[END_REF] , and illustrates a classical approach of expression proteomics. The synthetic image represents a 2D gel profile of rat kidney proteins, and the few proteins which amount is altered are indicated with arrows. The direction of the arrow indicates the direction and intensity of the change (decrease or increase), while its length indicates the statistical significance of the change. Among the six proteins showing the most significant changes, three were identified with the technical means available at that time (Edman microsequencing). Spot 75 was identified as Calbindin D, spot 96 as Regucalcin, and spot 109 as Major Urinary Protein.

Since this start toxicoproteomics has flourished in almost all possible directions and for all possible biological systems, from bacteria to plants going through all classes of animals. It would be overwhelming to review all these various aspects of toxicoproteomics, so that choices were made in preparing this paper. In order to select the cited references, Medline was searched with the keywords "toxicoproteomics" and "toxicity + proteomics". Only the papers dealing with mammalian cells and tissues were then kept for inclusion in this review, without any further selection on the type of toxicant or on the proteomic platform used. So despite their obvious interest, nothing will be found here about the responses of plants or bacteria to metals or to organic pollutants, nor will be anything mentioned about the field of ecotoxicology (reviewed for example in [START_REF] Dowling | Proteomics as a route to identification of toxicity targets in environmental toxicology[END_REF]) nor about biological fluids.

Even in this limited frame, toxicoproteomics has attracted a strong interest, and has been regularly reviewed (e.g. [START_REF] Steiner | Expression profiling in toxicology -potentials and limitations[END_REF][START_REF] Burchiel | Analysis of genetic and epigenetic mechanisms of toxicity: Potential roles of toxicogenomics and proteomics in toxicology[END_REF][START_REF] Moller | Two-dimensional gel electrophoresis: a powerful method to elucidate cellular responses to toxic compounds[END_REF][START_REF] Walgren | Application of proteomic technologies in the drug development process[END_REF][START_REF] Wetmore | Toxicoproteomics: Proteomics applied to toxicology and pathology[END_REF][START_REF] Collins | Use of proteomics for the discovery of early markers of drug toxicity[END_REF][START_REF] Merrick | The role of toxicoproteomics in assessing organ specific toxicity[END_REF][START_REF] Gao | Quantitative proteomics for drug toxicity[END_REF][START_REF] George | Toxicoproteomics: New paradigms in toxicology research[END_REF][START_REF] Amacher | The discovery and development of proteomic safety biomarkers for the detection of drug-induced liver toxicity[END_REF][START_REF] Schmitz-Spanke | Protein expression profiling in chemical carcinogenesis: a proteomic-based approach[END_REF][START_REF] Raida | Drug target deconvolution by chemical proteomics[END_REF][START_REF] George | Pesticides and cancer: insights into toxicoproteomicbased findings[END_REF][START_REF] Van Summeren | Proteomics in the search for mechanisms and biomarkers of drug-induced hepatotoxicity[END_REF][START_REF] Hess | The emerging field of chemo-and pharmacoproteomics[END_REF], to quote just a few). However, several of these reviews have either focused into specialized topics in toxicoproteomics (e.g. on polyaromatic hydrocarbons [START_REF] Verma | Review on proteomic analyses of benzo[a]pyrene toxicity[END_REF]), or have diluted proteomics with the other omics in the field of toxicology [START_REF] Aardema | Toxicology and genetic toxicology in the new era of "toxicogenomics": impact of "-omics" technologies[END_REF][START_REF] Merrick | Complementary gene and protein expression studies and integrative approaches in toxicogenomics[END_REF][START_REF] Harrill | Systems biology and functional genomics approaches for the identification of cellular responses to drug toxicity[END_REF] . It is thus felt that a review encompassing the various types of proteomics in the field of human-oriented toxicology can be of interest. In this context, two major types of proteomic studies in the field of toxicology can be distinguished: those aiming at studying the molecular responses in a biological system exposed to a toxicant and those aiming at finding directly the molecular targets of a toxicant.

Proteomics for studying biological responses to toxicants

The rationale of this approach is that examination of the biological responses will provide valuable insights into the molecular mechanisms at play when a cell or an organism is exposed to a given toxicant (Figure 2). Thus, the general implementations of this approach are to submit the cell or organism either to a moderately toxic (sub-lethal) dose of the chemical of interest or to a highly toxic (lethal) dose but to examine the target cell or tissue at early time points, where the cellular death has not increased too much. In both cases, the rationale is to use a high enough dose to induce a significant cellular response that can be easily observed by proteomics, keeping at the same time the cell mortality low enough not to get the proteomic analysis polluted by events that are strictly related to cell death and will not give interesting insights into the specific molecular mechanisms involved in toxic injury. The uppercase S at the end of sampleS stresses the importance of biological replicates for each condition. It should also be kept in mind that all expression proteomic strategies rely on quantitative differences, so that the quality of quantification and especially the variance of the quantification is crucial in such approaches. The steps downstream the proteomic process per se are also highlighted, e.g. the homeostasis hypothesis, which implies that what changes is important in the biological process studied, and the necessity of functional validation to derive solid biological knowledge from the proteomic results

The proteomes of the control and treated samples are then compared to determine which proteins are modulated (underexpressed or overexpressed) in the treated samples compared to the control ones. This statement implies that the proteomic setup used to perform this task must be quantitative. The number of biological replicates required to obtained reliable data also implies that the precision of the quantification must be kept over large series of samples. These features have a strong impact on the type of proteomic technology that is used to perform such studies. The responses that emerge from this type of toxicoproteomic analyses are a mixture of direct and indirect responses. The direct responses concern the target(s) of the toxicant. They can show up either in a decrease of the target protein(s), e.g. when the toxicant chemically modifies it and drive the protein(s) into degradation, e.g. by the proteasome pathway, or in an increase of the target proteins, by which the cell reacts to the toxicant by an increased synthesis of the target protein(s) to overcome the molecular inhibition caused by the toxicant. These direct responses however, are usually diluted by much wider indirect responses by which the cell/tissue tries to restore its homeostasis that has been compromised by the toxicant. These indirect responses are also of interest, as (i) they provide clues to the molecular pathways that are disturbed by the toxicant, even if the primary targets are not identified, and (ii) also give clues to molecular pathways that the cells upregulate/downregulate to survive, providing testable hypotheses on how helping the cells to survive (or oppositely increase their death rate) by chemically modulating these pathways. This frame of indirect toxicoproteomics is clearly the most widely used up to now, and it has been applied for all major types of toxicants.

Toxicoproteomics of responses to drugs

Adverse reactions are a major cause of drug withdrawal, either at the non-clinical stage or even worse at the clinical stage [START_REF] Lazarou | Incidence of adverse drug reactions in hospitalized patients: a meta-analysis of prospective studies[END_REF]. It is therefore of great importance to understand these toxic effects for several reasons: (i) it could enable the researchers to devise new leads keeping the efficacy and decreasing the toxic side effects (ii) according to the "fail early principle" the faster the toxic effect can be detected and even better predicted, the better it is in the drug development process.

As the toxic effects can arise through many different molecular mechanisms, it is therefore not surprising that the wide scope of proteomics has been used to detect and understand the involved mechanisms. The studies have focused on two types of drugs. First, the drugs that has been withdrawn from the market despite their efficacy because of strong adverse reactions (e.g. troglitazone) or which have shown important and potentially severe side effects (e.g. acetaminophen, cyclosporine, statins). Second, the anticancer drugs, for which toxicity is a concern for normal cells but is wanted for the cancer cells. In this respect, decoding the often more-complex-than-anticipated toxic mechanisms is of great importance to understand how to devise more efficient anticancer drugs with less resistances. *the proteomic setups are defined as follows : 2D gels : non multiplexed 2D gels, where proteins are detected by Coomassie staining, silver staining, fluorescence or radioactivity DIGE : multiplexed 2D gels with several fluorophores (one different per sample) MALDI : protein identification my MALDI-MS (peptide mass fingerprining) with or without an added MS/MS stage LCMS : protein identification by nanoLC-MS/MS techniques Shotgun spectral counting : label-free and 2D gels-free proteomics in which the proteins are empirically quantified through the spectral counting of their independent peptides appearing in the MS/MS analysis Shotgun label-free : label-free and 2D gels-free proteomics in which the proteins are empirically quantified by the measurement of the ion current corresponding to their peptides.

Examples of such toxicoproteomic studies on drugs are given in

The data compiled in Table 1 deal with studies that investigate mostly the effect of a single drug, sometimes two [START_REF] Lamoureux | Quantitative proteomic analysis of cyclosporine-induced toxicity in a human kidney cell line and comparison with tacrolimus[END_REF][START_REF] Groebe | Unexpected common mechanistic pathways for embryotoxicity of warfarin and lovastatin[END_REF]. However, some studies investigate the toxicity of a combination of drugs, either of the same therapeutic class (e.g. non steroidal anti-inflammatory drugs on rat cardiomyocytes using a 2D gel approach [START_REF] Baek | Proteomic analysis in NSAIDs-treated primary cardiomyocytes[END_REF]) or of different classes but with the same target organ, such as the nephrotoxicity study carried out in [START_REF] Matheis | Cross-study and cross-omics comparisons of three nephrotoxic compounds reveal mechanistic insights and new candidate biomarkers[END_REF] using 2D DIGE or the hepatotoxicity studies carried out either using 2D gels [START_REF] Man | Protein expression analysis of drug-mediated hepatotoxicity in the Sprague-Dawley rat[END_REF][START_REF] Thome-Kromer | Toward the identification of liver toxicity markers: A proteome study in human cell culture and rats[END_REF], or 2D DIGE [START_REF] Van Summeren | Proteomics Investigations of Drug-Induced Hepatotoxicity in HepG2 Cells[END_REF]. Series of studies addressing the same topic with different experimental designs (e.g. [START_REF] Man | Protein expression analysis of drug-mediated hepatotoxicity in the Sprague-Dawley rat[END_REF][START_REF] Thome-Kromer | Toward the identification of liver toxicity markers: A proteome study in human cell culture and rats[END_REF][START_REF] Van Summeren | Proteomics Investigations of Drug-Induced Hepatotoxicity in HepG2 Cells[END_REF]) are very important on a general point of view, as they allow assessing the relevance and specificity of the proteomic findings while removing the experimenter and technical setup variables. Such studies indeed allowed demonstrating that different drugs induce different responses. Conversely, comparison of studies made on similar drugs with the same proteomic setup and the same tissues, e.g. [START_REF] Steiner | Cholesterol biosynthesis regulation and protein changes in rat liver following treatment with fluvastatin[END_REF][START_REF] Steiner | Proteomics to display lovastatin-induced protein and pathway regulation in rat liver[END_REF] show similar cellular responses. However, when taken globally, some pathways and some proteins are found rather often, highlighting potential roles in response to injury or in detoxification processes. For example, calreticulin is found as a responsive protein in 7 studies made on different toxicants [START_REF] Fountoulakis | Twodimensional database of mouse liver proteins: Changes in hepatic protein levels following treatment with acetaminophen or its nontoxic regioisomer 3acetamidophenol[END_REF][START_REF] Gamberi | Proteomic analysis of A2780/S ovarian cancer cell response to the cytotoxic organogold(III) compound Aubipy[END_REF][START_REF] Tian | Azacytidine induces necrosis of multiple myeloma cells through oxidative stress[END_REF][START_REF] Chavez | Quantitative proteomic and interaction network analysis of cisplatin resistance in HeLa cells[END_REF][START_REF] Groebe | Unexpected common mechanistic pathways for embryotoxicity of warfarin and lovastatin[END_REF][START_REF] Park | Proteomic analysis to identify early molecular targets of pregabalin in C6 glial cells[END_REF][START_REF] Man | Protein expression analysis of drug-mediated hepatotoxicity in the Sprague-Dawley rat[END_REF]. Mitochondrial proteins (albeit different ones) are almost always found, chaperones of the grp family are found in 10 studies [START_REF] Fountoulakis | Twodimensional database of mouse liver proteins: Changes in hepatic protein levels following treatment with acetaminophen or its nontoxic regioisomer 3acetamidophenol[END_REF][START_REF] Uttenweiler-Joseph | Quantitative proteomic analysis to decipher the differential apoptotic response of bortezomib-treated APL cells before and after retinoic acid differentiation reveals involvement of protein toxicity mechanisms[END_REF][START_REF] Lamoureux | Quantitative proteomic analysis of cyclosporine-induced toxicity in a human kidney cell line and comparison with tacrolimus[END_REF][START_REF] Dukes | Changes in endoplasmic reticulum stress proteins and aldolase A in cells exposed to dopamine[END_REF][START_REF] Cecconi | Proteomic analysis of rat cortical neurons after fluoxetine treatment[END_REF][START_REF] Zhang | Proteomic changes of PC12 cells treated with proteasomal inhibitor PSI[END_REF][START_REF] Koncarevic | Differential genomic and proteomic profiling of glioblastoma cells exposed to terpyridineplatinum(II) complexes[END_REF][START_REF] Maniratanachote | Chaperone proteins involved in troglitazone-induced toxicity in human hepatoma cell lines[END_REF][START_REF] Man | Protein expression analysis of drug-mediated hepatotoxicity in the Sprague-Dawley rat[END_REF][START_REF] Van Summeren | Proteomics Investigations of Drug-Induced Hepatotoxicity in HepG2 Cells[END_REF], and proteasome subunits in 10 studies [34, 36, 37, 39, 45, 53-55, 57, 63], just to quote a few. It is noteworthy that P450 cytochromes are only found in a few studies using shotgun-type proteomics, [START_REF] Cho | In-depth Identification of Pathways Related to Cisplatin-induced Hepatotoxicity through an Integrative Method Based on an Informatics-assisted Label-free Protein Quantitation and Microarray Gene Expression Approach[END_REF][START_REF] Cho | Integrative analysis of proteomic and transcriptomic data for identification of pathways related to simvastatin-induced hepatotoxicity[END_REF][START_REF] Cho | Proteome profiling of tolbutamide-treated rat primary hepatocytes using nano LC-MS/MS and label-free protein quantitation[END_REF], as they are not visualized by 2D electrophoresis. However, they are not seen in most of the shotgun-type proteomic studies either. A few additional studies can be related to this topic of drugs toxicity, namely the studies that investigate either the interference of natural products with drug effects (e.g. with doxorubicin in [START_REF] Chen | Quercetin-induced cardioprotection against doxorubicin cytotoxicity[END_REF] and in [START_REF] Diaz-Chavez | Proteomic profiling reveals that resveratrol inhibits HSP27 expression and sensitizes breast cancer cells to doxorubicin therapy[END_REF]) or the studies that investigate resistance to drugs, especially resistance to anticancer drugs (e.g. [START_REF] Lincet | Comparative 2D-DIGE proteomic analysis of ovarian carcinoma cells: toward a reorientation of biosynthesis pathways associated with acquired platinum resistance[END_REF][START_REF] Murphy | Proteomic investigation of taxol and taxotere resistance and invasiveness in a squamous lung carcinoma cell line[END_REF][START_REF] Urbani | A proteomic investigation into etoposide chemo-resistance of neuroblastoma cell lines[END_REF][START_REF] Qi | Overexpression of sorcin in multidrug resistant human leukemia cells and its role in regulating cell apoptosis[END_REF][START_REF] Di Michele | A proteomic approach to paclitaxel chemoresistance in ovarian cancer cell lines[END_REF][START_REF] Peng | A proteomic investigation into adriamycin chemo-resistance of human leukemia K562 cells[END_REF][START_REF] Brown | Investigation of doxorubicin resistance in MCF-7 breast cancer cells using shot-gun comparative proteomics with proteolytic O-18 labeling[END_REF][START_REF] Sinha | Study of the development of chemoresistance in melanoma cell lines using proteome analysis[END_REF][START_REF] Castagna | A proteomic approach to cisplatin resistance in the cervix squamous cell carcinoma cell line A431[END_REF]). This subject is of old interest for the proteomic community, as can be seen from reviews that are older than a decade, e.g. [START_REF] Hutter | Proteomics for studying cancer cells and the development of chemoresistance[END_REF], and research papers that are of course even older [START_REF] Sinha | Increased expression of annexin I and thioredoxin detected by two-dimensional gel electrophoresis of drug resistant human stomach cancer cells[END_REF][START_REF] Sinha | Increased expression of epidermal fatty acid binding protein, cofilin, and 14-3-3-sigma (stratifin) detected by two-dimensional gel electrophoresis, mass spectrometry and microsequencing of drug-resistant human adenocarcinoma of the pancreas[END_REF].

Toxicoproteomics of responses to natural products

Owing to the fact that many drugs derive from natural products, the boundary between toxicoproteomics of drugs and of natural products is fuzzy and somewhat arbitrary. Despite the fact that the financial incentive is much less for this area of research than for drugs, an important number of toxicoproteomic papers has been published in this area, and a selection of the mis displayed in table 2. In vitro (human) Hela 2D gels/MALDI [START_REF] Xu | Tubeimoside-1 Exerts Cytotoxicity in HeLa Cells through Mitochondrial Dysfunction and Endoplasmic Reticulum Stress Pathways[END_REF] usnic acid

In vivo (rat) liver 2D gels/MALDI [START_REF] Liu | Proteomic Study on Usnic-Acid-Induced Hepatotoxicity in Rats[END_REF] When the data that are compiled in this table are cross-analyzed, the emerging keyword is ER stress, where the key players are the ER chaperones. ER stress is mentioned in 8 of the 17 entries of Table 2 ([82,[START_REF] Hansen | Quantitative Proteomics Reveals Cellular Targets of Celastrol[END_REF][START_REF] Siu | Proteomic and transcriptomic study on the action of a cytotoxic saponin (Polyphyllin D): Induction of endoplasmic reticulum stress and mitochondria-mediated apoptotic pathways[END_REF][START_REF] Short | Apoptosis induced by staurosporine alters chaperone and endoplasmic reticulum proteins: Identification by quantitative proteornics[END_REF][START_REF] Pan | Proteomic analysis reveals tanshinone IIA enhances apoptosis of advanced cervix carcinoma CaSki cells through mitochondria intrinsic and endoplasmic reticulum stress pathways[END_REF][START_REF] Foldi | Proteome-wide study of endoplasmic reticulum stress induced by thapsigargin in N2a neuroblastoma cells[END_REF][START_REF] Xu | Tubeimoside-1 Exerts Cytotoxicity in HeLa Cells through Mitochondrial Dysfunction and Endoplasmic Reticulum Stress Pathways[END_REF][START_REF] Liu | Proteomic Study on Usnic-Acid-Induced Hepatotoxicity in Rats[END_REF]). The second keyword is mitochondria, mitochondrial proteins being found modulated in 7 of the 17 entries of Table 2 ([80,[START_REF] Fung | Butyrate-Induced Apoptosis in HCT116 Colorectal Cancer Cells Includes Induction of a Cell Stress Response[END_REF][START_REF] Feng | ER stress-mediated apoptosis induced by celastrol in cancer cells and important role of glycogen synthase kinase-3 beta in the signal network[END_REF][START_REF] Hansen | Quantitative Proteomics Reveals Cellular Targets of Celastrol[END_REF][START_REF] Wang | Proteomic characterization of the possible molecular targets of pyrrolizidine alkaloid isoline-induced hepatotoxicity[END_REF][START_REF] Heo | Toxicoproteomic analysis of phalloidin-induced cholestasis in mouse liver[END_REF][START_REF] Foldi | Proteome-wide study of endoplasmic reticulum stress induced by thapsigargin in N2a neuroblastoma cells[END_REF] ). This suggests that the mitochondrial response and the ER stress response are core cellular responses to which many different toxic mechanisms converge, and which may represent, together with oxidative stress and central metabolism [START_REF] Wang | Generally detected proteins in comparative proteomics -A matter of cellular stress response?[END_REF], a type of core response to stress and to cellular injury.

Toxicoproteomics of reponses to industrial chemicals

Another large class of toxicants that has been submitted to toxicoproteomic studies is represented by industrial chemicals, which can induce various toxic effects upon exposure. Various classes have been investigated, with of course a more intense focus on chemical classes that are known to represent a major public health concern, such as aromartic hydrocarbons or endocrine disruptors. These studies are summarized on table 3

Polychloro biphenyls

In vivo (rat) brain DIGE/MALDI [START_REF] Kodavanti | Aroclor 1254, a developmental neurotoxicant, alters energy metabolism-and intracellular signaling-associated protein networks in rat cerebellum and hippocampus[END_REF] Polychloro biphenyls

In vivo (rat) brain Shotgun (label free) [START_REF] Campagna | Cerebellum proteomics addressing the cognitive deficit of rats perinatally exposed to the food-relevant polychlorinated biphenyl 138[END_REF] Polychloro biphenyls

In vitro (rat) neurons Shotgun (spectral counting) [START_REF] Brunelli | Insight into the neuroproteomics effects of the food-contaminant non-dioxin like polychlorinated biphenyls[END_REF] thioacetamide In vivo (rat) liver 2D gels/MALDI [START_REF] Low | A proteomic analysis of thioacetamide-induced hepatotoxicity and cirrhosis in rat livers[END_REF] toluene In vitro (human) Lung carcinoma DIGE/MALDI [START_REF] Murugesan | Subtoxic and toxic concentrations of benzene and toluene induce Nrf2-mediated antioxidative stress response and affect the central carbon metabolism in lung epithelial cells A549[END_REF] It must be emphasized that some topics, such as the toxicity of polycyclic aromatic hydrocarbons, have raised an important interest that is reflected in the proteomic community, e.g. by a recent review on this specific topic [START_REF] Verma | Review on proteomic analyses of benzo[a]pyrene toxicity[END_REF]. This review goes beyond the two papers that have been compiled in Table 3 and addresses topics such as the use of reactive metabolites of benzopyrenes and/or chemoprevention of the carcinogenic effects of polycyclic aromatic hydrocarbons by natural products. As in Table 1, Table 3 compiles studies that investigate the effect of one chemical, or sometimes of a few related ones [START_REF] Murugesan | Subtoxic and toxic concentrations of benzene and toluene induce Nrf2-mediated antioxidative stress response and affect the central carbon metabolism in lung epithelial cells A549[END_REF][START_REF] Morbt | Chlorinated benzenes cause concomitantly oxidative stress and induction of apoptotic markers in lung epithelial cells (A549) at nonacute toxic concentrations[END_REF][START_REF] Brunelli | Insight into the neuroproteomics effects of the food-contaminant non-dioxin like polychlorinated biphenyls[END_REF]. However, very few studies are available where several different chemicals have been investigated in parallel. One exception is a study on various polycyclic aromatic hydrocarbons and related products such as coal tar [START_REF] Hooven | Proteomic analysis of MCF-7 cells treated with benzo[a]pyrene, dibenzo[a,l]pyrene, coal tar extract, and diesel exhaust extract[END_REF]. Conversely, some studies introduce an extra focus and apply proteomic technologies either to investigate a small subset of proteins such as the proteasome subunits [START_REF] Yuan | Proteomic profiling of expression of proteasomal subunits from livers of mice treated with diethylnitrosamine[END_REF] or to target proteins bearing a given posttranslational modification such as tyrosine phosphorylation [START_REF] De Graauw | Proteomic analysis of alternative protein tyrosine phosphorylation in 1,2-dichlorovinyl-cysteineinduced cytotoxicity in primary cultured rat renal proximal tubular cells[END_REF]. From Table 3, it can be easily seen that toxicoproteomic studies are focused on a few classes of chemicals (e.g. aromatic hydrocarbons) showing long-term effects. For these chemicals, where there are no obvious short-term effects in in vitro or on animal models, deciphering early mechanisms of action through omics technologies should enable the prediction a long-term outcome and would be of great value for effective and fast toxicological assessment. However, it must be recalled that this goal has not been reached yet. As previously discussed, some proteins/subsets of proteins are found at a high frequency in this area of toxicoproteomics of industrial products, illustrating their central role in response to toxic injury. HSP proteins (mostly hsp70 and hsp27) are found in 14 papers out of 30 ([99, 101-103, 105, 107, 112, 113, 116, 117, 120, 122, 123, 127]). Mitochondrial proteins, most often matricial enzymes, are also found in 14 papers out of 30 ([98, 99, 106, 107, 113, 114, 116-118, 120, 122, 123, 125, 126]). More surprisingly, the single protein enolase is found in 13 papers out of 30 ([98-101, 104, 105, 107, 109, 112, 116, 118, 125, 127]). Peroxiredoxins are also found, frequently, in 10 papers out of 30 ([98, 99, 102, 107, 110-112, 116, 119, 121]) and proteasome subunits in also 10 papers out of 30 ([99-101, 105, 107, 110, 111, 114, 125, 128]). Furthermore, as the toxicoproteomic studies on industrial chemicals are carried out on a limited range of chemicals, it is possible (and tempting) to compare various studies carried out on the same chemical in different papers (e.g. [START_REF] Oh | Effects of benzo(a)pyrene on protein expression in Jurkat T-cells[END_REF] vs [START_REF] Verma | Benzo[a]pyrenemediated toxicity in primary pig bladder epithelial cells: A proteomic approach[END_REF] on benzopyrene, [START_REF] Betancourt | Altered carcinogenesis and proteome in mammary glands of rats after prepubertal exposures to the hormonally active chemicals bisphenol a and genistein[END_REF] vs [START_REF] Lee | Analysis of differentially regulated proteins in TM4 cells treated with bisphenol A[END_REF] on bisphenol A, [START_REF] Shen | Differential expression of peroxiredoxin 6, annexin A5 and ubiquitin carboxyl-terminal hydrolase isozyme L1 in testis of rat fetuses after maternal exposure to di-n-butyl phthalate[END_REF] vs [START_REF] Zhang | Differential expression of peroxiredoxin 6 in fetal rat testis following in utero exposure to di(nbuty1) phthalate[END_REF] on dibutylphthalate, and [START_REF] Sarioglu | Quantitative analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced proteome alterations in 5L rat hepatoma cells using isotope-coded protein labels[END_REF] vs. [START_REF] Uchida | Proteome analysis of the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on murine testicular Leydig and Sertoli cells[END_REF] on dioxin). These comparisons show that proteomic studies carried out on the same chemical by different groups usually share a moderate overlap in the lists of modulated proteins. However, such comparisons should be handled with care, as the variation of the identified proteins depends on several factors. The first is of course biological variability, i.e. the fact that different cells may have a different response. The second factor is a purely technical one, i.e. the depth at which the proteome is investigated. Even keeping with studies that use the same proteomic setup, e.g. 2D gels, it is obvious that a 2D gel showing 2000 spots will show protein variations that will not appear on gels showing only 500-600 spots. In addition to these two obvious factors, a third subtler one must be taken into account, and relates to the way by which significant changes are detected in proteomic experiments. Significant changes are detected by statistical tests, so that the coefficient of variation observed for each protein in each condition plays a key role in defining the significance of a variation. Thus, if for any biological/technical reason the CV observed on protein X is higher in study B than in study A, then the same low-fold difference may be found significant (and thus reported) in study A, while it may be found not significant (and thus not reported) in study B.

Toxicoproteomics of reponses to metals and metalloids

Apart from the few metal complexes that are used as drugs (mostly platinum complexes and gold complexes), toxicology of metals, metalloids and their organic complexes is a branch of occupational toxicology. This does not prevent toxicoproteomics to participate to the general study of inorganics toxicology, as can be shown in the studies mentioned in Table 4. However, a review recently published on toxicoproteomics for the investigation of metal toxicity [START_REF] Luque-Garcia | Proteomics as a tool for examining the toxicity of heavy metals[END_REF] clearly indicates that in this specific area, toxicoproteomics on plants, microorganisms and metazoans of ecotoxicological interest dominates the field.

HeLa

2D gels [START_REF] Rousselet | Zinc adaptation and resistance to cadmium toxicity in mammalian cells: molecular insight by proteomic analysis[END_REF] The proteomic responses observed with metals, metalloids and their organic derivatives are similar to the ones observed with small organic molecules (Table 3). Heat shock proteins are found modulated in more than half of the papers ( [START_REF] Berglund | Proteomic analysis of low dose arsenic and ionizing radiation exposure on keratinocytes[END_REF][START_REF] Singhto | Proteome changes in human monocytes upon interaction with calcium oxalate monohydrate crystals[END_REF][START_REF] Singhto | Alterations in macrophage cellular proteome induced by calcium oxalate crystals: the association of HSP90 and F-actin is important for phagosome formation[END_REF][START_REF] Zhang | Dihydrolipoamide dehydrogenase and cAMP are associated with cadmium-mediated Leydig cell damage[END_REF][START_REF] Lei | Proteomic analysis of chromium cytotoxicity in cultured rat lung epithelial cells[END_REF][START_REF] Raghunathan | Changes in protein expression associated with chronic in vitro exposure of hexavalent chromium to osteoblasts and monocytes: A proteomic approach[END_REF][START_REF] Zhang | Heavy metals chromium and neodymium reduced phosphorylation level of heat shock protein 27 in human keratinocytes[END_REF][START_REF] Han | Proteomic analysis of the copper ion-induced stress response in a human embryonic carcinoma cell line[END_REF][START_REF] Cuello | Differential protein expression of hepatic cells associated with MeHg exposure: deepening into the molecular mechanisms of toxicity[END_REF][START_REF] Pan | Skin toxicology of lead species evaluated by their permeability and proteomic profiles: A comparison of organic and inorganic lead[END_REF][START_REF] Prat | Transcriptomic and proteomic responses of human renal HEK293 cells to uranium toxicity[END_REF]). Enolase is also found modulated frequently ([132, 133, 135, 139, 142, 144, 147, 151]), as well as ER stress ( [START_REF] Berglund | Proteomic analysis of low dose arsenic and ionizing radiation exposure on keratinocytes[END_REF][START_REF] Thongboonkerd | Proteomic Analysis of Calcium Oxalate Monohydrate Crystal-Induced Cytotoxicity in Distal Renal Tubular Cells[END_REF][START_REF] Zhang | Dihydrolipoamide dehydrogenase and cAMP are associated with cadmium-mediated Leydig cell damage[END_REF][START_REF] Lei | Proteomic analysis of chromium cytotoxicity in cultured rat lung epithelial cells[END_REF][START_REF] Petrak | Proteomic analysis of iron overload in human hepatoma cells[END_REF][START_REF] Cuello | Differential protein expression of hepatic cells associated with MeHg exposure: deepening into the molecular mechanisms of toxicity[END_REF]), proteasome ( [132, 135, 136, 145, 147, 148] ), or peroxiredoxins ( [START_REF] Thongboonkerd | Proteomic Analysis of Calcium Oxalate Monohydrate Crystal-Induced Cytotoxicity in Distal Renal Tubular Cells[END_REF][START_REF] Singhto | Alterations in macrophage cellular proteome induced by calcium oxalate crystals: the association of HSP90 and F-actin is important for phagosome formation[END_REF][START_REF] Choi | Genomic and Proteomic Profiling of the Cadmium Cytotoxic Response in Human Lung Epithelial Cells[END_REF][START_REF] Zhang | Dihydrolipoamide dehydrogenase and cAMP are associated with cadmium-mediated Leydig cell damage[END_REF][START_REF] Cuello | Differential protein expression of hepatic cells associated with MeHg exposure: deepening into the molecular mechanisms of toxicity[END_REF] ). It must be emphasized that peroxiredoxins are found in all the papers mentioned in Table 4 and dealing with strongly thiophilic metals such as mercury or cadmium, which is consistent with their thiol-containing active site.

Toxicoproteomics of reponses to nanoobjects

Nanoobjects, mostly nanoparticles and nanofibers, are more and more a potential concern for human health, and are therefore under intense scrutiny for possible adverse health effects. As it is difficult to forecast the behavior of nanoobjects in biological environments and their interaction with living cells, toxicoproteomics is almost ideal in this field to figure out which cellular responses are at play when cells encounter nanoobjects. It is therefore not surprising that toxicoproteomics studies have developed in this field, as shown in Table 5 Table 2D gels/MALDI [START_REF] Karlsson | Wear Particles from Studded Tires and Granite Pavement Induce Pro-inflammatory Alterations in Human Monocyte-Derived Macrophages: A Proteomic Study[END_REF] In this area, the results of toxicoproteomic studies are much less constant and consistent than in the previous sections. Sometimes usual players are found, such as the ER stress markers [START_REF] Tsai | Identification of the Nanogold Particle-Induced Endoplasmic Reticulum Stress by Omic Techniques and Systems Biology Analysis[END_REF][START_REF] Yuan | Comparative protein profile of human hepatoma HepG2 cells treated with graphene and single-walled carbon nanotubes: an iTRAQ-coupled 2D LC-MS/MS proteome analysis[END_REF], proteasome subunits ( [START_REF] Haniu | Proteomics-based safety evaluation of multi-walled carbon nanotubes[END_REF][START_REF] Cha | Proteomic identification of macrophage migration-inhibitory factor upon exposure to TiO2 particles[END_REF][START_REF] Triboulet | Analysis of cellular responses of macrophages to zinc ions and zinc oxide nanoparticles: a combined targeted and proteomic approach[END_REF][START_REF] Karlsson | Wear Particles from Studded Tires and Granite Pavement Induce Pro-inflammatory Alterations in Human Monocyte-Derived Macrophages: A Proteomic Study[END_REF]) or peroxiredoxins ( [155, 160, 161, 164] ). In other cases none of these classical proteins are found [START_REF] Okoturo-Evans | Elucidation of Toxicity Pathways in Lung Epithelial Cells Induced by Silicon Dioxide Nanoparticles[END_REF][START_REF] Jeon | Proteomic profiling of the differentially expressed proteins by TiO2 nanoparticles in mouse kidney[END_REF], which can be related to the weak toxicity of the precise nanoobject in the tested biological system. Nonetheless, all the toxicoproteomic studies carried out on nanoobjects have provided new mechanisms well beyond the oxidative stress that is almost always searched for in this area. Such mechanisms include interference with the central metabolism for ZnO nanoparticles, [START_REF] Triboulet | Analysis of cellular responses of macrophages to zinc ions and zinc oxide nanoparticles: a combined targeted and proteomic approach[END_REF], with glutathione metabolism and mitochondria for Cu and CuO nanoparticles [START_REF] Triboulet | Molecular Responses of Mouse Macrophages to Copper and Copper Oxide Nanoparticles Inferred from Proteomic Analyses[END_REF], or the more classical unfolded protein response [START_REF] Tsai | Identification of the Nanogold Particle-Induced Endoplasmic Reticulum Stress by Omic Techniques and Systems Biology Analysis[END_REF][START_REF] Okoturo-Evans | Elucidation of Toxicity Pathways in Lung Epithelial Cells Induced by Silicon Dioxide Nanoparticles[END_REF] for silica and nanogold, respectively.

Proteomics for finding molecular targets of toxicants

Because of its wide scope and depth of analysis, proteomics can be happily used to identify molecular targets of toxicants, which is one of the major stakes in toxicology.

Compared to the study on cellular responses, the name of the game is no longer to compare series of biological samples treated with different doses of a toxicant, but to analyze a few samples in which the molecular targets of the toxicant will have been made selectively detectable. Depending on the type of interaction between the targets to be found and the toxicant, i.e. covalent vs. non-covalent interactions, very different experimental schemes are used.

Finding targets by non-covalent interactions: chemical proteomics

In this type of studies, a very classical affinity chromatography setup is usually implemented (Figure 3). The molecule of interest is immobilized on a solid phase, on which a cell or tissue lysate prepared under non-denaturing conditions is loaded. After interaction and washes to remove the non-specifically bound proteins, elution is performed either by protein denaturation (thorough but less specific) or competition with a soluble form of the bait (lower yields but more specific) and the released proteins are analyzed by proteomic techniques. The purpose of a chemical proteomic is to identify the proteins that bind to a pharmacophore of interest. For technical reasons this pharmacophore is bound to a handle (clickable chemical group or directly a solid bead) via a linker (probe A). The handle allows retrieving selectively the proteins bound to the pharmacophore from the complex biological extract, and the linker is necessary to decrease the interferences between the pharmacophore and the handle. However, some proteins can bind to the linker or to the handle, and thus can be unspecifically retrieved. To alleviate this problem common to all affinity-based proteomic approaches, a negative control (probe B) must be used in parallel to substract the background proteins.

Examples of this approach are compiled in Table 6. As can be seen, the hot topic in this area is to find drug targets (known targets and off targets), and this new field has been already reviewed [START_REF] Rix | Target profiling of small molecules by chemical proteomics[END_REF][START_REF] Bantscheff | Chemoproteomic approaches to drug target identification and drug profiling[END_REF][START_REF] Colinge | Systems biology analysis of protein-drug interactions[END_REF]. It is clear from Table 6 that shotgun proteomics is used for finding drug targets while 2D gels are applied to find targets of metals. There is however an exception to this rule [START_REF] Duncan | An unbiased evaluation of CK2 inhibitors by chemoproteomics: characterization of inhibitor effects on CK2 and identification of novel inhibitor targets[END_REF], in which the binding screen is not a positive one (small molecule of interest bound to the resin) but a negative one (pharmacological inhibitor in solution and competition with the natural ligand for the target bound to the resin). This general trend is due to the widely different affinities of the two classes of molecules for their targets. Affinity of drugs for their targets, even their off-targets, is usually very high, in the low submicromolar range. This means in turn that in an affinity chromatography screen, strong washes can be implemented to remove all the contaminants before the final elution of the targeted proteins. In this frame, shotgun proteomics excels by its exquisite sensitvity. In the case of metals, the situation is very different. The affinity of proteins for metals is usually quite poor. This affinity must not be confused with the affinity of metalloproteins for their metals, which can be very high, e.g. in the zinc finger proteins. However, metalloproteins with such strongly bound metals are not demetallated when they are prepared before to binding to the immobilized metal, so that the affinity chromatography will really reveal the binding to proteins of excess metal, which is the case looked at in toxicoproteomics of metals. Thus, with such low affinities, excessive washes on the column would result into excessive losses of metal-binding proteins. This means in turn that the eluates of immobilized metal columns usually contain a non-negligible proportion of background proteins. In this frame, 2D gels bring their unique ability of an intra sample quantitative analysis, something that shotgun proteomics does not easily perform. 2D gels also bring a very easy comparison between the eluate and the various controls that are needed to find the metal-binding proteins in this experimental setup. It must also be kept in mind that affinity chromatography is not the ideal situation for finding targets. First, the binding of the affinity probe to the bead may introduce steric constraints that prevent some protein targets from binding to the column. A classical example in the non-drug field is represented by the quest for ATP-binding proteins by affinity chromatography. Immobilization of the ATP by the base is the easiest but also the most-interfering one, as the protein must differentiate ATP from the other NTPs in the cell and therefore "read" the base moiety of ATP [START_REF] Haystead | Gammaphosphate-linked ATP-sepharose for the affinity purification of protein kinases. Rapid purification to homogeneity of skeletal muscle mitogen-activated protein kinase kinase[END_REF]. Thus, immobilization of ATP by the terminal phosphate with spacer arms must be used to improve the representation of the ATP-binding proteome [START_REF] Ito | Analysis of the soluble ATP-binding proteome of plant mitochondria identifies new proteins and nucleotide triphosphate interactions within the matrix[END_REF][START_REF] Lee | Identification of differentially expressed proteins by treatment with PUGNAc in 3T3-L1 adipocytes through analysis of ATP-binding proteome[END_REF] . Transposed to the chemical proteomic field, this means that a good knowledge of the binding of at least some target proteins must be earned prior to designing the affinity adsorbent. Second, and in addition to this problem, affinity chromatography is usually performed on whole lysates. This means for example that some of the proteins that will see the bait in the affinity chromatography setup would never be in contact with the drug in vivo because of the cellular compartmentalization. In addition, the binding assay will be performed under a single condition in terms of ionic strength and buffer composition (e.g. presence or absence of divalent cations) and this precise condition may not be appropriate for all targets. This means that schemes in which the interaction of the bait with the proteins are made under in vivo conditions, following the principles developed in B. Cravatt's group [START_REF] Speers | Chemical strategies for activity-based proteomics[END_REF], prior to final immobilization of the bait-target complexes, e.g. by click chemistry [START_REF] Speers | Activity-based protein profiling in vivo using a copper(i)-catalyzed azide-alkyne [3 + 2] cycloaddition[END_REF] or avidin chromatography [START_REF] Liu | Activity-based protein profiling: the serine hydrolases[END_REF], are usually more performing than classical affinity chromatography setups for finding drug targets [START_REF] Shi | Cell-based proteome profiling of potential dasatinib targets by use of affinity-based probes[END_REF].

Targets of chemicals determined by covalent binding

Covalent binding of chemicals to biomolecules is also a very important toxic mechanism. Besides the obvious binding of electrophilic chemicals to the various nucleophilic sites present in biomolecules (e.g. bases in nucleic acids, lysine and cysteine side chains in proteins), it is also known for decades that chemicals that are not electrophilic per se, such as aromatic hydrocarbons, can undergo in vivo an oxidative metabolism that can produce electrophilic species such as epoxides [START_REF] Jerina | The role of arene oxide-oxepin systems in the metabolism of aromatic substrates. 3. Formation of 1,2-naphthalene oxide from naphthalene by liver microsomes[END_REF], which are the real causative agents of the toxic phenomena observed, such as necrosis [START_REF] Brodie | Possible mechanism of liver necrosis caused by aromatic organic compounds[END_REF] or tumorigenesis [START_REF] Grover | In vitro transformation of rodent cells by K-region derivatives of polycyclic hydrocarbons[END_REF]. This bioactivation process is not restricted to aromatic hydrocarbons and can happen for many molecules (e.g. in [START_REF] Mitchell | The role of biotransformation in chemical-induced liver injury[END_REF]). It is of course worth to be investigated in order to determine how toxicity arises when such reactive metabolites are produced, and to identify their molecular targets. In the protein world, the fact that the core technologies used in proteomics have the potential to detect these targets is known since even before the word proteomics was even coined [START_REF] Anderson | Covalent protein modifications and gene expression changes in rodent liver following administration of methapyrilene: a study using two-dimensional electrophoresis[END_REF][START_REF] Richardson | Comparisons of Protein-Changes in Human and Rodent Hepatocytes Induced by the Rat-Specific Carcinogen, Methapyrilene[END_REF].

In this type of toxicoproteomic analysis, the name of the game is to detect and identify the proteins that have bound the metabolites of the chemicals of interest. In an ideal world, the analysis should go down to the modified peptide, so that the modified site can be identified. This is however not often possible, as the chemical adduction often alters strongly the physico-chemical properties of the modified peptides. Murphy's law being what it is, this means that the modified peptides are often very difficult to obtain and identify. First, the exact adducting entity must be known to be able to select the modified peptides in the mass spectrometer, and this is often uneasy. Second, adduction on cysteines is not a real problem for the production and extraction of the modified peptides, but adduction on lysines is a problem, as it causes an automatic miscleavage with trypsin, sometimes with a charge loss, so that extracting and finding the modified peptide becomes almost impossible. Shotgun proteomics approaches have been described to identify modified peptides following exposure to reactive chemicals. However, the biological relevance of the data generated from such large screens needs to be cautiously evaluated [START_REF] Martyniuk | Protein targets of acrylamide adduct formation in cultured rat dopaminergic cells[END_REF][START_REF] Ariza | Protein haptenation by amoxicillin: high resolution mass spectrometry analysis and identification of target proteins in serum[END_REF] Thus, is often much safer to stop at the modified protein level, the bulk of the unmodified residues in the protein acting as a buffer to keep everything soluble and amenable to analysis (Figure 4). can be taken to alleviate this problem when the modified peptide is not detected.

In this frame, the modified proteins can sometimes be detected just by the pI shift on 2D gels [START_REF] Anderson | Covalent protein modifications and gene expression changes in rodent liver following administration of methapyrilene: a study using two-dimensional electrophoresis[END_REF][START_REF] Richardson | Comparisons of Protein-Changes in Human and Rodent Hepatocytes Induced by the Rat-Specific Carcinogen, Methapyrilene[END_REF]. When the metabolite is large enough to be used as an hapten, detection of the modified proteins by antibodies is also feasible [START_REF] Asif | Proteins identified as targets of the acyl glucuronide metabolite of mycophenolic acid in kidney tissue from mycophenolate mofetil treated rats[END_REF]. However, in most of the cases, the chemical is given as a radioactive precursor and the modified proteins are detected through detection of radioactivity. Positional alignment on gels where a similar but nonradioactive extract has been loaded allows picking up the proteins for identification by standard techniques. This rather cumbersome setup is required by the fact that it is not possible to chemically modify the molecule to be investigated, as this would result into an altered metabolism. There are therefore very few options to detect the molecule of interest when bound to the target proteins.

Examples of this strategy are given in Table 7 below

Shotgun

Mass adduct [START_REF] Martyniuk | Protein targets of acrylamide adduct formation in cultured rat dopaminergic cells[END_REF] bromobenzene Rat liver (in vivo)

22D gels/MALDI radioactivity [START_REF] Koen | A proteomic analysis of bromobenzene reactive metabolite targets in rat liver cytosol in vivo[END_REF] bromophenol Rat liver (in vivo)

2D gels/MALDI radioactivity [START_REF] Koen | Liver protein targets of hepatotoxic 4-bromophenol metabolites[END_REF] dopamine Human neuroblastoma (in vitro)

2D gels/MALDI radioactivity [START_REF] Van Laar | Proteomic identification of dopamine-conjugated proteins from isolated rat brain mitochondria and SH-SY5Y cells[END_REF] furan Rat liver (in vivo)

2D gels/LCMS radioactivity [START_REF] Moro | Identification and Pathway Mapping of Furan Target Proteins Reveal Mitochondrial Energy Production and Redox Regulation as Critical Targets of Furan Toxicity[END_REF] monocrotaline Human endothelial cells(in vitro)

2D gels/MALDI radioactivity [START_REF] Lame | Monocrotaline pyrrole targets proteins with and without cysteine residues in the cytosol and membranes of human pulmonary artery endothelial cells[END_REF] mycophenolat e

Rat kidney (in vivo)

2D gels/LCMS blotting [START_REF] Asif | Proteins identified as targets of the acyl glucuronide metabolite of mycophenolic acid in kidney tissue from mycophenolate mofetil treated rats[END_REF] naphthalene Mouse liver microsomes (in vitro) 2D gels/MALDI radioactivity [START_REF] Isbell | Identification of proteins adducted by reactive naphthalene metabolites in vitro[END_REF] quinones Human bronchial cells(in vitro)

2D gels/MALDI radioactivity [START_REF] Lame | Protein targets of 1,4benzoquinone and 1,4-naphthoquinone in human bronchial epithelial cells[END_REF] thioacetamide Rat hepatocytes (in vitro)

2D gels/LCMS radioactivity [START_REF] Koen | Protein targets of thioacetamide metabolites in rat hepatocytes[END_REF] thiobenzamide Rat liver (in vivo)

2D gels/MALDI radioactivity [START_REF] Ikehata | Protein targets of reactive metabolites of thiobenzamide in rat liver in vivo[END_REF] This strategy builds in an important caveat, however, which is the comigration problem in 2D gels [START_REF] Campostrini | Spot overlapping in two-dimensional maps: A serious problem ignored for much too long[END_REF]. This comigration problem is not always a major issue in classical proteomics [START_REF] Hunsucker | Is protein overlap in two-dimensional gels a serious practical problem?[END_REF], but in this precise toxicoproteomic frame, the real target protein can be a minor component and not the major one. As the modified peptide is often not detected, there is always a doubt in the assignment of the target proteins. Thus, the implicit gamble that is made in the studies cited in Table 7 is that the reactivity of all proteins present within the same 2D spot is similar, so that the most abundant one is the real target. In the absence of functional validation (e.g. showing that the activity of the identified protein is lower when the cells have been treated with the target chemical) this gamble appears rather risky. There are however means to address the comigration problem, either by using a third separation [START_REF] Colignon | Evaluation of threedimensional gel electrophoresis to improve quantitative profiling of complex proteomes[END_REF], or by using narrow pH gradients in which the comigration probability becomes negligible [START_REF] Hoving | Towards high performance twodimensional gel electrophoresis using ultrazoom gels[END_REF], [START_REF] Westbrook | Zooming-in on the proteome: very narrow-range immobilised pH gradients reveal more protein species and isoforms[END_REF]. However, all these strategies require a second round of experiments, which is not very practical in setups that are already rather slow because of the time needed to detect the radioactive signal. Thus, the optimal strategy in this case is probably to use directly multiple narrowrange pH gradients in the first round of experiments. Although requiring more experiments and more sample at once, the time saved by carrying out only one round of long experiments instead of two is probably worth the effort. Once a given target protein has been identified, specific proteomic investigations can sometimes be carried out to characterize the modified residues. Reliable MS-based protocols have been recently described for this purpose [START_REF] Zhang | Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation[END_REF][START_REF] Peng | Optimizing proteolytic digestion conditions for the analysis of serum albumin adducts of 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine, a potential human carcinogen formed in cooked meat[END_REF] Compared to the situation of chemical proteomics in the previous section, the specificity issues in the case of covalent binding do not arise from the general experimental setup, as everything is made in vivo with the required molecule only, but arise from the readout system that is used.

Indirect targets

For some toxicants, generic toxic mechanisms such as oxidative stress are implied.

In such cases, one possible aim of toxicoproteomic studies is to find the targets of the reactive oxidant species produced during the oxidative stress. These targets are identified through the chemical modifications induced by the oxidative stress, and the modifications most commonly used are tyrosine nitration and protein carbonylation, which are identified by immunoblotting either directly (tyrosine nitration) or after derivatization with a suitable hydrazine used as a hapten (protein carbonylation).

Only a few examples are present in the toxicoproteomic literature, one on tyrosine nitration [START_REF] Fan | Anilineinduced nitrosative stress in rat spleen: Proteomic identification of nitrated proteins[END_REF], and two on protein carbonylation [START_REF] Bruno | Protein Carbonyl Formation in Response to Propiconazole-induced Oxidative Stress[END_REF][START_REF] Steiner | A comparative study of protein carbonylation and mitochondrial dysfunction using the neurotoxicants 1,3dinitrobenzene, 3-nitropropionic acid, and 3-chloropropanediol[END_REF].

The rather low popularity of these studies can be explained by the fact that they require blotting and gel alignment steps that make them more cumbersome to carry out that classical response-type studies. In addition, and oppositely to what is encountered in other types of target research, they often yield indirect targets, which is not felt as interesting as direct targets.

A critical analysis of the achievements of toxicoproteomics

With almost 20 years of toxicoproteomics and several tens of papers published in the field, it is now possible to make a critical appraisal of the literature, which gives quite interesting insights regarding the technical choices and the value of the results

Technical issues in toxicoproteomics

On a technical point of view, there is a strong overrepresentation of 2D gel-based proteomics in toxicoproteomics compared to other areas of proteomics in cell biology. This question arises in the toxicoproteomics of cell responses and not really in the targets finding area, where there is a clear-cut distinction between non covalent interactions (dominated by shotgun proteomics) and covalent binding (dominated by 2D gels). This overrepresentation of 2D gels may be linked in part to the historical perspective inherent to a review paper. However, as most of the papers cited here are less than five years old, this preeminence of 2D gels is likely to be linked to their higher reproducibility when large series of samples are analyzed. The high reproducibility of 2D gels has been demonstrated over the years , with median coefficient of variations in the 20-30% range [START_REF] Merkley | Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes[END_REF][START_REF] Maes | Interindividual variation in the proteome of human peripheral blood mononuclear cells[END_REF][START_REF] Winkler | Biological variation of the platelet proteome in the elderly population and its implication for biomarker research[END_REF], even in large sample series. Such coefficient of variations have only recently been accessible to shotgun proteomics. For label-free methods, there are still limited to either short series of experiments [START_REF] Gautier | Label-free quantification and shotgun analysis of complex proteomes by one-dimensional SDS-PAGE/NanoLC-MS: evaluation for the large scale analysis of inflammatory human endothelial cells[END_REF], or to a moderate depth of analysis [START_REF] Wang | Comprehensive comparison of iTRAQ and label-free LC-based quantitative proteomics approaches using two Chlamydomonas reinhardtii strains of interest for biofuels engineering[END_REF]. In methods using stable isotope labelling, the intraexperiment coefficients of variations are low but limited to the multiplex ability of the method [START_REF] Wang | Comprehensive comparison of iTRAQ and label-free LC-based quantitative proteomics approaches using two Chlamydomonas reinhardtii strains of interest for biofuels engineering[END_REF][START_REF] Uitto | Comparing SILAC and two-dimensional gel electrophoresis image analysis for profiling urokinase plasminogen activator signaling in ovarian cancer cells[END_REF], thereby limiting the number of samples that can be analyzed. Moreover, the missing value problem is still a major issue in shotgun proteomics [START_REF] Antberg | Critical comparison of multidimensional separation methods for increasing protein expression coverage[END_REF], so that sophisticated and still questionable imputation strategies must be implemented [START_REF] Teeguarden | Comparative Proteomics and Pulmonary Toxicity of Instilled Single-Walled Carbon Nanotubes, Crocidolite Asbestos, and Ultrafine Carbon Black in Mice[END_REF]. In addition, deriving a variation in a protein amount from several variations at the peptide level is still a difficult issue [START_REF] Karp | Addressing accuracy and precision issues in iTRAQ quantitation[END_REF]. Conversely, 2D gels are able to handle similar sample series with high reproducibility [START_REF] Triboulet | Analysis of cellular responses of macrophages to zinc ions and zinc oxide nanoparticles: a combined targeted and proteomic approach[END_REF] provided that a modern image analysis software is used [START_REF] Stessl | Influence of image-analysis software on quantitation of two-dimensional gel electrophoresis data[END_REF].

This high sensitivity to reproducibility issues may be specifically increased in the context of toxicoproteomics. First, many toxicoproteomic studies are carried out on animal tissues after an in vivo exposure. As animal tissues are much more variable than pure in vitro models, adding a lot of technical variability on top of this biological variability is likely to decrease strongly the power of the study. Second, even for in vitro models and as shown in the very basic dose-survival curves presented in Figure 5, the reproducibility is excellent when the cells are all alive or all dead. In the middle, and especially in the high slope zone around the EC50, the experimental variability increases dramatically. As most of the toxicoproteomic studies are carried out between EC10 and EC50 in order to visualize an effect, there are also carried out in this zone of high biological variability. In this context, adding a high technical variability to this already high biological variability would just blur, statistically speaking, the effects to be observed. However, the reproducibility afforded by 2D gels come at a price, which is the limited depth of analysis of the proteomes. It is therefore not surprising that most of the toxicoproteomic studies published to date converge on shared mechanisms dealing with rather abundant proteins.

The results hierarchization issue

Apart from this technical point, toxicoproteomic studies, as all proteomic studies, suffer from a major concern that roots in the lack of hierarchization intrinsic to the approach. Taking the example of the interaction of small molecules with their protein targets, classical biochemical studies provide a list of Kd together with the list of proteins, helping to sort which proteins will bind the molecule first. When transposed to a proteomic setup, in this case a chemical proteomic one, all proteins with a low enough Kd to keep the binding throughout the experiment will show up exactly the same way and with no hierarchization at all. This problem is not limited to chemical proteomics. In the case of cellular biology, any cellular response is a mixture of essential responses (essential meaning here required to survive the toxic challenge) and of "fitness" responses (responses increasing the performance of the cells but not mandatory to survive). Proteomic experiments do not bring any information in sorting the essential responses from the fitness ones, while this information is much needed to derive biologically relevant conclusions.

In this respect, it is tempting to sort the proteomic results by the significance index provide by the statistical tests (maximal fold change and or minimal risk value). However, it should be kept in mind that the extent of a change in terms of induction or repression fold is not necessarily correlated with its importance in cell survival processes. For example, making 20% more of an already abundant protein may represent a major effort for a cell and be important for its survival.

There is also a tendency to consider as important what is specific and as unimportant what is frequently encountered in the response to different toxicants. Here again there is no evidence backing this rule, and oppositely a commonly encountered response may well be common exactly because it is useful to increase survival under several circumstances.

The validation issue

Thus, the only way to differentiate essential responses from non-essential ones is to perform followup, validation experiments that will aim not at confirming the biochemical changes already seen in proteomics, but at determining how the changes observed in the proteomic screen correlate or not, experimentally speaking, with the cell survival or cell damage upon the toxic challenge. However, in toxicoproteomics as well as in proteomics in general, studies embedding their own functional validation experiments are uncommon, to say the least. Nevertheless, it is quite informative to dissect what concepts and experiments have been used to carry out functional validation.

When an enzyme appears as modulated in a proteomic screen, there are two possible levels of validation. The first one is to measure the intracellular levels of the products of the enzymes, in order to check whether the observed change in the enzyme level correlates with a change in activity. This approach has been carried out in the case of A549 cells treated with benzene and toluene [START_REF] Murugesan | Subtoxic and toxic concentrations of benzene and toluene induce Nrf2-mediated antioxidative stress response and affect the central carbon metabolism in lung epithelial cells A549[END_REF]. Several proteins involved in the central metabolism were modulated in response to these aromatic hydrocarbons, and the authors measured the levels of several metabolites to get a better picture of the real metabolic alterations going on. With the development of metabolomic techniques, such approaches should become even easier and greatly informative. In the same trend, enzyme activities can be measured to confirm that changes observed in the proteomic screen do translate in the same changes in the enzyme activity, and are not just attempts of compensation of a toxicant-induced decrease in activity. Such approaches have also been described for metabolic enzymes [START_REF] Vanamala | Resveratrol suppresses human colon cancer cell proliferation and induces apoptosis via targeting the pentose phosphate and the talin-FAK signaling pathways-A proteomic approach[END_REF][START_REF] Triboulet | Analysis of cellular responses of macrophages to zinc ions and zinc oxide nanoparticles: a combined targeted and proteomic approach[END_REF]. When possible, an even more interesting validation, on a toxicological point of view, is to feed the cells challenged with the toxicant with the product of this enzyme. There are a few examples of this approach in toxicoproteomic studies. When HL60 leukemia cells are treated with arsenite, a down regulation of fatty acid synthase is observed [START_REF] Xiong | Quantitative proteomic analysis reveals the perturbation of multiple cellular pathways in HL-60 cells induced by arsenite treatment[END_REF]. Feeding the arsenite-treated cells with palmitic acid increased cell survival, hereby showing that at least part of the arsenite toxicity was due to the inhibition of fatty acid synthesis.

In the same trend, it has been shown that several central metabolism enzymes are modulated in macrophage cells treated with zinc [START_REF] Triboulet | Analysis of cellular responses of macrophages to zinc ions and zinc oxide nanoparticles: a combined targeted and proteomic approach[END_REF] or in ovary cancer cells treated with a gold complex [START_REF] Gamberi | Proteomic analysis of A2780/S ovarian cancer cell response to the cytotoxic organogold(III) compound Aubipy[END_REF]. Feeding the cells with extra pyruvate, the end product of the glycolysis and pentose phosphate pathways, increases cell survival upon zinc or organogold challenge, respectively. Maybe one of the most exquisite examples based on activity validation is also one of the oldest toxicoproteomic piece of work. In a series of papers, S. Steiner's group showed first that one of the major effects of cyclosporine A on kidney was to decrease the level of calbindin [START_REF] Steiner | Cyclosporine A decreases the protein level of the calcium-binding protein calbindin-D 28kDa in rat kidney[END_REF]. They subsequently showed that this decrease was translated into impaired calcium repumping in the kidney, leading to calcium mineralization in the renal tubules [START_REF] Aicher | New insights into cyclosporine A nephrotoxicity by proteome analysis[END_REF][START_REF] Aicher | Decrease in kidney calbindin-D 28kDa as a possible mechanism mediating cyclosporine A-and FK-506induced calciuria and tubular mineralization[END_REF].

Another interesting path for validation studies is to use pharmacological inhibitors (or, more rarely, stimulators) of the pathways that have been evidenced in the proteomic screen. In some instances, the rationale is to add a pharmacological inhibition on top of the inhibition observed with the toxicant tested, and to check whether this results in a higher mortality. An example of this approach can be found with proteasome inhibition in response to zinc [START_REF] Triboulet | Analysis of cellular responses of macrophages to zinc ions and zinc oxide nanoparticles: a combined targeted and proteomic approach[END_REF].

In other cases, the pharmacological inhibitor is used to counter the cellular response and to check the effects on cell survival. When macrophage cells are treated with copper oxide nanoparticles, both the glutathione biosynthesis and the mitochondrial respiratory chain are up-regulated [START_REF] Triboulet | Molecular Responses of Mouse Macrophages to Copper and Copper Oxide Nanoparticles Inferred from Proteomic Analyses[END_REF]. Opposing these effects with non toxic concentrations of buthionine sulfoximine (an inhibitor of glutathione biosynthesis) or respiratory chain inhibitors, respectively, enhances the toxicity of the copper oxide nanoparticles. The same holds true with pharmacological inhibition of HSP90 with LiCl in celastrol-treated cells [START_REF] Feng | ER stress-mediated apoptosis induced by celastrol in cancer cells and important role of glycogen synthase kinase-3 beta in the signal network[END_REF], or with 17AAG in arsenic oxide-treated cells [START_REF] Ge | Proteomic and functional analyses reveal a dual molecular mechanism underlying arsenic-induced apoptosis in human multiple myeloma cells[END_REF].

ER stress has been described and put forward in many of the studies cited in this review. However, only two studies used the ER stress inhibitor salubrinal to check whether ER stress was instrumental in cell death/survival upon the toxic challenge or was just a side effect [START_REF] Feng | ER stress-mediated apoptosis induced by celastrol in cancer cells and important role of glycogen synthase kinase-3 beta in the signal network[END_REF][START_REF] Pan | Proteomic analysis reveals tanshinone IIA enhances apoptosis of advanced cervix carcinoma CaSki cells through mitochondria intrinsic and endoplasmic reticulum stress pathways[END_REF]. In both cases, treatment with salubrinal decreased cell death, showing the pivotal role of the ER stress in the toxic process.

Besides the use of pharmacological inhibitors, another way of manipulating the activity of a protein is to change its level through genetic engineering techniques, either by transfection (increase of the level) or more and more frequnetly by siRNA techniques (decrease of the level). Compared to chemical inhibitors, these techniques are less easy to implement but should work for each and every protein.

These techniques have seldom been used in toxicoproteomics, but their success rate is high. In most cases, these approaches target proteins that are upregulated in response to toxicants and that may represent survival factors. For example, the stress protein hsp60 is induced in response to digoxin [START_REF] Qiu | Comparative proteomics analysis reveals role of heat shock protein 60 in digoxin-induced toxicity in human endothelial cells[END_REF], and an increase of the level of this protein by transfection decreases the toxicity of digoxin. Conversely, Grp78 is induced in response to troglitazone [START_REF] Maniratanachote | Chaperone proteins involved in troglitazone-induced toxicity in human hepatoma cell lines[END_REF], and its forced decrease by siRNA increases the toxicity of troglitazone. The same dynamics holds true for valosincontaining protein in response to chromium [START_REF] Pan | Functional proteomics reveals hepatotoxicity and the molecular mechanisms of different forms of chromium delivered by skin administration[END_REF]. In a more subtle way, silencing of Hsp90 during treatment of macrophages with calcium oxalate interferes with the phagocytic activity of macrophages [START_REF] Singhto | Alterations in macrophage cellular proteome induced by calcium oxalate crystals: the association of HSP90 and F-actin is important for phagosome formation[END_REF]. There is also one example of a study targeting a protein that is decreased in response to a toxicant, namely the 14-3-3 zeta protein in response to arsenic trioxide [START_REF] Ge | Proteomic and functional analyses reveal a dual molecular mechanism underlying arsenic-induced apoptosis in human multiple myeloma cells[END_REF]. Quite interestingly, further decrease of the protein level by siRNA increases the sensitivity of the cells to the toxicant, while increasing the protein level by transfection decreased the action of the toxicant. This decrease of the 14-3-3 zeta protein when cells are exposed to arsenic trioxide is therefore very likely to be a direct effect of the toxicant, inducing a strong pro-death effect in the cells.

The specificity issue

In addition to this validation issue, there is another question that percolates the field of toxicoproteomics, which is the question of specificity. This question can be approached at two levels. The first level is to delineate the specific vs the generic responses in a given system. As shown in this review, the vast majority of the toxicoproteomic papers deal with a simple control vs toxicant-treated situation, so that the issue of specificity just cannot be addressed. Too few studies include "intermediary controls", i.e. toxicants of the same or of another chemical family, which is the only way to address the specificity issue. In the same trend, authors investigating serially a few toxicants in a series of papers usually do not use their previous results to address this issue.

The second level is more a problem of relevance. In other words, can a generic response or a response seeming unrelated to the process under investigation still be relevant?

The almost instinctive answer to this question is that when this happens, this means that proteomics unravels only the more visible, generic responses, and that the specific responses are not highlighted by the proteomic screen and remain undescribed. However, this intuitive view is sometimes wrong, as shown by the work on cell chemoresistance to anticancer drugs. For example, old toxicoproteomic work showed that cells resistant to drugs overexpress proteins such as cofilin [START_REF] Sinha | Increased expression of epidermal fatty acid binding protein, cofilin, and 14-3-3-sigma (stratifin) detected by two-dimensional gel electrophoresis, mass spectrometry and microsequencing of drug-resistant human adenocarcinoma of the pancreas[END_REF], while there is no obvious relation between resistance to drugs that induce DNA breaks and the overexpression of a cytoskeletal protein. Nevertheless, it was recently shown that simple cofilin overexpression confers resistance to cisplatin [START_REF] Becker | High cofilin-1 levels correlate with cisplatin resistance in lung adenocarcinomas[END_REF], of course by a yet completely unknown mechanism, but this validates the relevance of the proteomic work carried out 15 years ago. Besides cofilin, the same holds true for fatty acid binding proteins [START_REF] Sinha | Increased expression of epidermal fatty acid binding protein, cofilin, and 14-3-3-sigma (stratifin) detected by two-dimensional gel electrophoresis, mass spectrometry and microsequencing of drug-resistant human adenocarcinoma of the pancreas[END_REF][START_REF] Jeong | Fatty acid-binding protein 5 promotes cell proliferation and invasion in human intrahepatic cholangiocarcinoma[END_REF].

To conclude this section, it is a pity that so few proteomic papers in general and toxicoproteomic papers in particular include their own validation studies. Such an absence can be understood from ancient papers, e.g. from a decade ago, when the validation means were not what they are now. For more recent papers, with the analytical tools currently at hand and with the simplicity of the output to measure in the case of toxicoproteomic studies (i.e. cell death/survival), too many proteomic papers stop in the middle of the river, leaving the reader short of solid hypotheses or conclusions and just with speculations.

What can we think of shared/transversal mechanisms

Examination of the toxicoproteomic outcomes shows that some molecular mechanisms, such as metabolic activation (e.g. with enolase and other enzymes of the glycolytic pathway), ER stress, chaperones induction or mitochondrial stress are found in a high proportion of the toxicoproteomic papers, almost independently of the type of toxicant and thus probably independently of the initial mechanism of action. This phenomenon is reminiscent of the well-known déjàvu in proteomics, i.e. proteins which amounts is found to be modulated in many different experimental contexts [START_REF] Wang | Generally detected proteins in comparative proteomics -A matter of cellular stress response?[END_REF][START_REF] Petrak | Deja vu in proteomics. A hit parade of repeatedly identified differentially expressed proteins[END_REF]. It is therefore important to interpret it and figure out whether this is a core phenomenon or just a background effect. In order to perform this interpretation, we need to figure out what is going on in a cell exposed to a toxic. The toxicant will first distribute within the cell, but not uniformly, depending on its physico-chemical characteristics. For example, cationic species, whether inorganic cations of hydrophobic organic cations, are pumped in the mitochondria because of their transmembrane potential. Then the toxicant will bind to its molecular targets and inhibit them with several possible mechanisms. Because of the various feedback mechanisms present in cells, the cell will react on two initial levels:

(i) it will try to correct the molecular defect. If the target is DNA the cell will try to repair it, if the target is a protein the cell will synthesize some extra protein to restore the function (ii) it will try to remove the toxicant. This can be made by several mechanisms, such as pumping the molecule out of the cell, conjugating it to a biological acceptor such as glutathione to prevent its binding to the target (so that induction of glutathione transferases is commonly observed), or oxidizing it (e.g. with cytochromes P450 which are almost ubiquitously expressed [START_REF] Ding | Human extrahepatic cytochromes P450: function in xenobiotic metabolism and tissue-selective chemical toxicity in the respiratory and gastrointestinal tracts[END_REF]) here again to prevent it from binding. The point is that nothing comes for free in a cell, so that all of these processes call for extra energy, both redox energy (NADPH for the functioning of cytochromes P450 and of glutathione reductase) and chemical energy (NTPs to produce RNA and proteins). It is therefore no surprise to observe an induction of the energy-producing metabolism. This increased energy production comes at a price, however, which is an increased oxidative stress due to the incomplete reduction of oxygen. This oxidative stress is of course more important if energy production is one of the targets of the toxicant, i.e. a situation where the cell tries to activate a malfunctioning machinery. It is therefore no surprise either to see an induction of antioxidant proteins such as peroxiredoxins. Secondary to this oxidative stress but also to the structural destabilization that is sometimes induced by the toxicant, more proteins than usual get unfolded, so that a response to this accumulation of unfolded proteins is often induced, detected by an increase in the proteasome pathway and in the chaperones of various types. Last but not least, why are the ER and mitochondrial stress response so commonly activated? In other words, why are the ER and mitochondria more stressed than other parts of the cells? For the mitochondria, this is due both to their ability to pump various cationic species that become concentrated within the mitochondrial matrix, and to their sensitivity to oxidative stress if anything goes wrong with the energy metabolism, which is activated.

For the ER, the prevalence of the ER stress may be related to the fact that ER is also the site of localization of cytochromes P450. These molecules do degrade organic toxicants but they may release even more toxic products. In addition, for performing their oxidative chemistry cytochromes P450 activate the molecular oxygen into very reactive transient species. Normally these species are not released but any leak, even small, will create an intense oxidative stress and thus an unfolded protein response in the ER.

Thus, as well as for the classical déjà vu in proteomics [START_REF] Wang | Generally detected proteins in comparative proteomics -A matter of cellular stress response?[END_REF], the common mechanisms frequently encountered in toxicoproteomic correspond to core stress response mechanisms to exogenous toxicants, and they probably play a central role in the mechanisms that cell activate to survive the toxic stress. It is therefore not surprising that the few studies that have attempted to validate these aspects of cellular responses have succeeded and demonstrated their relevance to cell resistance to toxicants.

What future for toxicoproteomics ?

First of all, it should be mentioned that toxicoproteomics follows the rapid developement of proteomics in general, and almost two thirds of the papers cited in this review have been published in the last five years. This reflects the fact that proteomics is improving rapidly and has never been as good, fast and easy as now. More importantly, examination of the deliverables of the toxicoproteomic literature makes reasonable to state that proteomics is a sound approach in the field of toxicology, as it does deliver molecular targets and/or molecular mechanisms occurring in cells or tissues treated with toxicants, as shown by the papers presenting an experimental validation of the proteomic findings. This includes (but is not limited to) off target kinases [START_REF] Shi | Cell-based proteome profiling of potential dasatinib targets by use of affinity-based probes[END_REF], fatty acid synthase [START_REF] Xiong | Quantitative proteomic analysis reveals the perturbation of multiple cellular pathways in HL-60 cells induced by arsenite treatment[END_REF], glutathione biosynthesis [START_REF] Triboulet | Molecular Responses of Mouse Macrophages to Copper and Copper Oxide Nanoparticles Inferred from Proteomic Analyses[END_REF] or chaperones [START_REF] Maniratanachote | Chaperone proteins involved in troglitazone-induced toxicity in human hepatoma cell lines[END_REF][START_REF] Qiu | Comparative proteomics analysis reveals role of heat shock protein 60 in digoxin-induced toxicity in human endothelial cells[END_REF][START_REF] Ge | Proteomic and functional analyses reveal a dual molecular mechanism underlying arsenic-induced apoptosis in human multiple myeloma cells[END_REF][START_REF] Pan | Functional proteomics reveals hepatotoxicity and the molecular mechanisms of different forms of chromium delivered by skin administration[END_REF].

Despite these recent progresses, it can be reasonably stated that proteomics is strongly underused in mechanistic toxicology, although it has the strong advantage over transcriptomics to integrate all the phenomena that occur post transcriptionally and are an integral part of the biological response, e.g. chemical modifications, induced proteolytic degradation, impaired translation etc… There are multiple reasons to this rather limited use. First, even if becoming easier, proteomics is still perceived as cumbersome and very far from being a high throughput technique in the sense of toxicologists or pharmacologists. It should rather be seen as a high content technique that can deliver a lot of information, but on a limited set of conditions. Second, even in this specific frame, the question of the real impact of proteomics in toxicology should be raised. In the absence of functional validation, the impact is always limited. When the final output of a toxicoproteomic paper is just a list of proteins, and especially a list of modulated proteins instead of a list of direct targets, the reader is left in a difficult situation. Even when the list is short, it is usually full of proteins that do not show an obvious link with the anticipated action of the toxicant. And even if validation of these findings should be relatively easy, this distance to expectations is not tempting to carry out a validation, which burden should rest on the scientists having initiated the study. When the list of modulated proteins is very large, as afforded by transcriptomic or shotgun proteomic studies, additional pitfalls are encountered. First the lack of hierarchization in the results make it very difficult to decide where the validation effort should go in priority, given the fact that it will be impossible to validate so many protein changes. Second the multiple testing issue increases, and it has been shown that classical correction strategies often remove interesting changes and leave noise [START_REF] Diz | Multiple hypothesis testing in proteomics: a strategy for experimental work[END_REF]. Third, because of the missing value problem and of the difficulties in deriving a protein abundance change from several changes in peptide abundances (e.g. in [START_REF] Karp | Addressing accuracy and precision issues in iTRAQ quantitation[END_REF]) the robustness of the data decreases concomitantly with the increase in the number of proteins analyzed. Thus, the probability of having data that look statistically correct but are based on weak proteomic experimental evidences increases, and such data are bound to fail at the validation stage. All these reasons explain why proteomics has often been perceived as disappointing regarding its impact/investment ratio.

In addition, specificity will remain a problem because of the difficulty of analyzing very large series of samples with proteomic techniques, but the impact depends on the purpose of the study. When the goal is to find biomarkers [START_REF] Li | Biomarkers and human hepatocytes[END_REF][START_REF] Pal | Copper toxicity induced hepatocerebral and neurodegenerative diseases: An urgent need for prognostic biomarkers[END_REF] specificity is an issue, but it can be addressed by careful and ambitious study design, including not only a control and a single toxicant, but also several chemicals chosen to address the specificity issue (e.g. in [START_REF] Van Summeren | Proteomics in the search for mechanisms and biomarkers of drug-induced hepatotoxicity[END_REF][START_REF] Man | Protein expression analysis of drug-mediated hepatotoxicity in the Sprague-Dawley rat[END_REF][START_REF] Collins | Development of a Pharmaceutical Hepatotoxicity Biomarker Panel Using a Discovery to Targeted Proteomics Approach[END_REF]). However, specificity is not necessarily a condition for usefulness, and oppositely defining shared targets/reactions can be extremely useful in several areas.

One of these areas is the field of anticancer chemotherapy, where toxicoproteomics can be very useful. At the level of target finding, determining if different drugs have or not the same targets is a very valuable information for designing combined chemotherapies. At the level of cellular reactions, knowing how a cancer cell responds to a drug can give insights to counter this reaction and thus sensitize the cancer cell to the drug or decrease the side effects of the drug. Examples of this strategy are appearing in the literature [START_REF] Chen | Quercetin-induced cardioprotection against doxorubicin cytotoxicity[END_REF][START_REF] Diaz-Chavez | Proteomic profiling reveals that resveratrol inhibits HSP27 expression and sensitizes breast cancer cells to doxorubicin therapy[END_REF]. As another example, the fact that organogold compounds do induce a proteasome response [START_REF] Gamberi | Proteomic analysis of A2780/S ovarian cancer cell response to the cytotoxic organogold(III) compound Aubipy[END_REF] brings to the front the idea to combine organogold with proteasome inhibitors therapies. In this general frame, the goal of toxicoproteomic studies is to provide enough information to build combined chemotherapies based on molecular informations not only at the level of mode of action, but also at the level of cellular responses. The promise of such informed combined chemotherapies is to decrease dramatically the level of relapses and at the same time to decrease the toxic effects on healthy cells.

This conceptual frame will also be very useful in other areas of toxicology where toxicity is an unwanted event, whether occupational toxicology or drug toxicology. In fact, toxicoproteomics will be very useful to address one of the major challenges in modern toxicology, namely the toxicology of mixtures. Synergistic effects, either additive or through crosstalks, are a major issue, and addressing them requires a good molecular knowledge of the molecular effects of the toxicants, which proteomic can provide. For example, just by using knowledge already at hand, it would be interesting to verify if the completely different toxicants inducing ER stress show an additive toxicity, even if their primary mode of action is different. This concept can also be used at a finer level. In particular, knowing what a cell induces in response to a toxicant also tells where is becomes more vulnerable, and thus sensitive to other toxicants acting not additively, but through synergic mechanisms (e.g. celastrol and lithium as in [START_REF] Feng | ER stress-mediated apoptosis induced by celastrol in cancer cells and important role of glycogen synthase kinase-3 beta in the signal network[END_REF] ). Thus, toxiproteomics finds itself at the heart of predictive and mechanistic toxicology at two levels:

(i) by extracting as much information as possible from in vitro systems, it maximizes their usefulness and is part of the general move aiming at limiting the use of animals for toxicological screening (e.g. in [START_REF] Van Summeren | Proteomics in the search for mechanisms and biomarkers of drug-induced hepatotoxicity[END_REF][START_REF] Collins | Development of a Pharmaceutical Hepatotoxicity Biomarker Panel Using a Discovery to Targeted Proteomics Approach[END_REF]) (ii) by performing a screen of targets/responses without preconception, it helps at defining new molecular mechanisms and thus at making predictions about toxicity of combinations of chemicals, which is another sense of the word "predictive toxicology" Thus, it can be concluded that toxicoproteomics will be an important part of the roots from which the much-needed tree of toxicology, and especially toxicology of mixtures will grow, so that the future of the field should be bright. It will be even brighter if the toxicoproteomists take the good habit of pushing their studies to the end at all levels, as derived from the critical analysis outlined in section 4. On a proteomic technical point of view, it will mean to be very strict with the proteomic setups used. If 2D gels are preferred, they shall be pushed to their maximum in order to increase the depth of analysis and thus the probability to go beyond the usual players so often described in toxicoproteomics. If shotgun proteomics is preferred, special care should be taken to the statistical quality of the data. The analysis should be restricted to the proteins for which robust quantitative data can be derived, without resorting to imputation strategies to mask the missing values. Such poor data should be discarded without regrets, as there will stay more than enough proteins to be analyzed. On a more general point of view, no toxicoproteomic paper should appear without an experimental validation of the main findings, and the classical scheme of a binary comparison control vs treated with one toxicant should decrease in favor of more complex comparison including a non-treated sample and samples treated with several different products to address the specificity issue. Fulfilling these conditions will be the only way for proteomics to have a real impact in toxicology.

Figure 1 :

 1 Figure 1: Historical illustration of the interest of proteomics in toxicology This figure is extracted from one of the oldest toxicoproteomic papers[START_REF] Aicher | New insights into cyclosporine A nephrotoxicity by proteome analysis[END_REF] , and illustrates a classical approach of expression proteomics. The synthetic image represents a 2D gel profile of rat kidney proteins, and the few proteins which amount is altered are indicated with arrows. The direction of the arrow indicates the direction and intensity of the change (decrease or increase), while its length indicates the statistical significance of the change. Among the six proteins showing the most significant changes, three were identified with the technical means available at that time (Edman microsequencing). Spot 75 was identified as Calbindin D, spot 96 as Regucalcin, and spot 109 as Major Urinary Protein.

Figure 2 :

 2 Figure 2: Scheme of principle of expression proteomics This figure describes the major steps involved in an expression proteomic experiment.The uppercase S at the end of sampleS stresses the importance of biological replicates for each condition. It should also be kept in mind that all expression proteomic strategies rely on quantitative differences, so that the quality of quantification and especially the variance of the quantification is crucial in such approaches. The steps downstream the proteomic process per se are also highlighted, e.g. the homeostasis hypothesis, which implies that what changes is important in the biological process studied, and the necessity of functional validation to derive solid biological knowledge from the proteomic results

Figure 3 :

 3 Figure 3: Scheme of principle of chemical proteomics (non-covalent interactions)The purpose of a chemical proteomic is to identify the proteins that bind to a pharmacophore of interest. For technical reasons this pharmacophore is bound to a handle (clickable chemical group or directly a solid bead) via a linker (probe A). The handle allows retrieving selectively the proteins bound to the pharmacophore from the complex biological extract, and the linker is necessary to decrease the interferences between the pharmacophore and the handle. However, some proteins can bind to the linker or to the handle, and thus can be unspecifically retrieved. To alleviate this problem common to all affinity-based proteomic approaches, a negative control (probe B) must be used in parallel to substract the background proteins.

Figure 4 :

 4 Figure 4: Scheme of principle of covalent binding proteomicsThe purpose of the experiment is to identify the proteins that bind a chemical of interest or its reactive metabolites produced by bioactivation. The most classical, 2D gel-based process is illustrated on the figure. In this process the two critical steps are: (i)the correct matching of the labelled spots on the general 2D map. This can be made much easier by a low sensitivity, permanent staining prior to radioactivity detection (e.g. Coomassie blue staining) (ii) the handling of the multiple protein identifications from a single spot by mass spectrometry. Some measures (e.g. high resolution pH gradients in IEF, third dimension electrophoresis)

Figure 5 :

 5 Figure 5: variability of survival curves. J774 mouse macrophage cells are grown in 12-cell plates and exposed to zinc ion for 24 hours at the given concentration. Their viability after exposure is measured by the neutral red intake test. The same experiment is carried out in triplicate, and the results are displayed together on the same graph. The dispersion of the viability data in the EC20-EC75 range is easily seen, showing that cells exposed to a given concentration in replicate experiments can be in a fairly different physiological state.

Table

Table 1 :

 1 Toxicoproteomic studies on drugs

	Chemical	In	vivo/in	Organ/cell type Proteomic	reference
		vitro				setup*
	acetaminophenol	In	vivo	liver		2D	[33]
		(mouse)			gels/MALDI
	aminoacridines	In	vitro	various		2D	[34]
		(human)			gels/LCMS
	andrographolide	In	vitro	hepatocytes	Silac	[35]
		(human)
	aubipy	In	vitro	Ovary	tumor	2D	[36]
		(human)	cells		gels/MALDI
	aza cytidine	In	vitro	Pancreas		2D	[37]
		(human)	tumor cells	gels/MALDI
	aza cytidine	In	vitro	myeloma		TMT	[38]
		(human)
	bortezomib	In	vitro	myeloma		iTRAQ	[39]
		(human)
	cisplatin	In	vitro	Hela		Silac	[40]
		(human)
	cisplatin	In vitro (rat)	hepatocytes	Shotgun	[41]
						label free
	cisplatin	In	vitro	Ovary		Shotgun	[42]
		(human)	mitochondria	spectral
						count

Table 2 :

 2 Toxicoproteomics of natural products

	chemical	In vivo/in vitro	Organ/cell type Proteomic setup reference
	azaspiracid	In vitro (human) neuroblastoma silac	[80]
	bilirubin	In vitro (human) neuroblastoma	2D	[81]
					gels/MALDI
	butyrate	In vitro (human) Colon cancer	2D	[82]
			cells		gels/MALDI
	celastrol	In vitro (human) HeLa		2D	[83]
					gels/MALDI
	celastrol	In vitro (human) lymphoblast	silac	[84]
	deoxynivalenol In vitro (human) lymphocytes	2D	[85]
					gels/MALDI
	digoxin	in vitro (human) HUVEC		2D gels/LCMS [86]
	isoline	In vivo (mouse) liver		2D gels/LCMS [87]
	palytoxin	In vitro (human) Mammary		2D gels/LCMS [88]
			tumor cells
	phalloidin	In vivo (mouse) liver		2D	[89]
					gels/MALDI
	polyphyllin	In vitro (human) Various	cell	2D gels/MALDI [90]
			lines
	resveratrol	In vitro (human) Colon cancer	Shotgun label	[91]
			cells		free
	staurosporine	In vitro (human) neuroblastoma 2D gels/MALDI [92]
	tanshinone	In vitro (human) Cervix		2D	[93]
			carcinoma		gels/MALDI
	thapsigargin	In vitro (human) neuroblastoma 2D gels/LCMS [94]
	tubeimoside

Table 3 :

 3 Toxicoproteomic studies on industrial chemicals

	chemical	In vivo/in vitro Organ/cell type Proteomic setup reference
	acrolein	In vitro (rat) Lung	2D	[98]
				epithelium	gels/MALDI
	benzene	In	vitro	Lung	DIGE/MALDI [99]
		(human)	carcinoma
	benzopyrene	In	vitro	lymphoblast	2D	[100]
		(human)		gels/MALDI
	benzopyrene	In vitro (pig) urothelium	2D	[101]
					gels/MALDI
	bisphenol A	In vivo (rat)	Mammary	2D	[102]
				gland	gels/MALDI
	bisphenol A	In	vitro	Sertoli cell line 2D	[103]
		(mouse)			gels/MALDI
	bromate	In vivo (rat)	kidney	DIGE/LCMS	[104]
	Butylidene	In	vitro	Prostate	2D	[105]
	phthalide	(human)	cancer cells	gels/LCMS
	CCl4	In vivo (rat)	Liver	2D	[106]
				mitochondria	gels/MALDI
	chlorobenzenes	In	vitro	Lung	DIGE/MALDI [107]
		(human)	carcinoma
	decadienal	In	vitro	Bronchial cells 2D	[108]
		(human)		gels/LCMS
	diazinon	In	vitro	neuroblastoma 2D	[109]
		(mouse)			gels/MALDI
	dibutylphthalate	In vivo (rat)	Fetal testis	2D	[110]
					gels/MALDI
	dibutylphthalate	In vivo (rat)	Fetal testis	2D	[111]
					gels/MALDI
	diethylmaleate	In	vivo	Airway	DIGE/LCMS	[112]
		(mouse)		epithelium
	dimethoxyhexan	In vivo (rat)	testis	2D	[113]
	e				gels/MALDI
	dinitrobenzene	In vivo (rat)	testis	2D	[114]
					gels/MALDI
	dioxin (TCDD)	In	vitro	hepatoma	2D	[115]
		(human)		gels/MALDI
	dioxin (TCDD)	In vitro (rat) hepatoma	Shotgun	[116]
					(ICPL)
	dioxin (TCDD)	In	vitro	Leydig+sertoli	2D	[117]
		(mouse)		cell lines	gels/Edman
	fuel (alkanes)	In	vivo	lung	2D	[118]
		(mouse)			gels/MALDI
	Glyphosate	In	vivo	skin	2D	[119]
		(mouse)			gels/LCMS
	hydrazine	In vivo (rat)	liver	DIGE/MALDI [120]

Table 4 :

 4 Toxicoproteomic studies on metals and metalloids

	chemical	In vivo/in vitro	Organ/cell type Proteomic	reference
						setup
	arsenic trioxide I	In	vitro	myeloma	2D gels/MALDI [131]
		(human)
	arsenite	In		vitro	myeloma	SILAC	[132]
		(human)
	arsenite	In		vitro	keratinocytes	2D gels/MALDI [133]
		(human)
	diphenylarsinic	In		vitro	Hepatoma cell	2D gels/MALDI [134]
	acid	(human)		line
	calcium	In vitro (dog)	Kidney cell line 2D gels/MALDI [135]
	oxalate
	calcium	In		vitro	Monocyte cell	2D gels/LCMS [136]
	oxalate	(human)		line
	calcium	In		vitro	Macrophage	2D gels/LCMS [137]
	oxalate	(human)		cell line

Table 6 :

 6 Detection of protein targets of chemicals through non-covalent interaction

	Chemical	Biological		Affinity type	Proteomic	Reference
		system			setup
	abl inhibitors	HeLa cells		immobilized	ITRAQ	[172]
	abl inhibitors	K562 cells		immobilized	shotgun	[173]
	andrographolid	Various human	In solution	ITRAQ	[174]
	e	cell lines
	bosutinib	Various human	immobilized	shotgun	[175]
		cell lines
	CK2 inhibitors	HeLa cells		Immobilized	2D gels/MALDI [176]
				(indirect)
	dasatinib	Various human	Immobilized	shotgun	[177]
		cell lines		vs. In solution
	E-3180	Ovary cancer	immobilized	SILAC	[178]
		cell line
	gelfitinib	Human		immobilized	SILAC	[179]
		myeloma cell
		line
	HDAC inhibitors Various human	immobilized	ITRAQ & TMT [180]
		cell lines
	INNO-406	Human		immobilized	shotgun	[181]
		leukemia and
		normal blood
		cells
	PBT-1	Human	lung	immobilized	shotgun	[182]
		cancer	cell
		lines
	copper and zinc Various human	immobilized	2D gels/LCMS [183]
		hepatoma cell
		lines
	copper	human		immobilized	2D gels/MALDI [184]
		hepatoma cell
		line
	zinc	Mouse		immobilized	2D gels/LCMS [167]
		macrophage
		cell line

Table 7 :

 7 Detection of protein targets of chemicals through covalent labelling

	Chemical	Target cell	Proteomic	Detection	reference
			setup	method
	acrolein	Mouse heart	2D gels/MALDI blotting	[200]
		(in vivo)
	acrylamide	Rat neurons(in
		vitro)

Acknowledgments

The authors have declared no conflict of interest. TR thanks the CEA-toxicology program for financial support.