Lemasson Alban
email: alban.lemasson@univ-rennes1.fr

Lippi Daria
email: daria.lippi@fabriqueautonome.org

Hamelin Laura
email: hamelin_laura@orange.fr

Louazon Stéphane
email: stephane.louazon@univ-rennes1.fr

Hausberger Martine
email: martine.hausberger@univ-rennes1.fr

Public attitude influences actors' visual orientation: a pilot experimental study

Human emotions guide verbal and non-verbal behaviour during social encounters. During public performances, performers' emotions can be affected directly by an audience's attitude. The valence of the emotional state (positive or negative) of a broad range of animal species is known to be associated with a body and visual orientation laterality bias. Here, we evaluated the influence of an audience's attitude on professional actors' head orientation and gaze direction during two theatrical performances with controlled observers' reactions (Hostile vs Friendly audience). First, our speech fluency analysis confirmed that an audience's attitude influenced

actors' emotions. Second, we found that, whereas actors oriented more their head to the left (i.e. Right Hemisphere Bias) when the audience was hostile, they gazed more straight ahead at Friendly spectators. These results are in accordance with the Valence-Specific Hypothesis that proposes that processing stimuli with negative valences involves the right hemisphere (i.e. left eye) more than the left hemisphere.

KEYWORDS

INTRODUCTION

Nonverbal behaviours, such as mutual gazes and proxemics, impact the regulation of social interactions [START_REF] Herrera | Dialog Behaviors across Culture and Group Size, in: Universal Access in Human-Computer Interaction[END_REF]. Directed gazes and body postures play an important role in coordinating turn-taking by communicating information concerning interlocutors' intentions and emotions [START_REF] Kendon | Some functions of gaze-direction in social interaction[END_REF][START_REF] Herrera | Dialog Behaviors across Culture and Group Size, in: Universal Access in Human-Computer Interaction[END_REF]. Body posture affects an audience's perception [START_REF] Huang | Powerful Postures Versus Powerful Roles: Which Is the Proximate Correlate of Thought and Behavior?[END_REF], but an audience's characteristics can also influence performers' body posture [START_REF] Mehrabian | Significance of posture and position in the communication of attitude and status relationships[END_REF]. Emotions and behaviours are intermingled [START_REF] Feldman | Negative emotions and acute physiological responses to stress[END_REF][START_REF] Baumeister | How Emotion Shapes Behavior: Feedback, Anticipation, and Reflection, Rather Than Direct Causation[END_REF]. Gaze avoidance is characteristic of unfriendly encounters, while more direct gazes are present during friendly interactions [START_REF] Kendon | Some functions of gaze-direction in social interaction[END_REF]. Interlocutors also tend to expose one hemiface more than the other to their partner [START_REF] Nicholls | The effect of left and right poses on the expression of facial emotion[END_REF]. The choice of the hemiface exposed is linked to the subject's emotional state (i.e. presenting the left cheek when posing is considered to be more emotionally expressive, [START_REF] Nicholls | Laterality of expression in portraiture: putting your best cheek forward[END_REF]2002) or to the emotional valence (aggressive/friendly) of the context of social encounters [START_REF] Basile | Social and Emotional Values of Sounds Influence Human (Homo sapiens) and Non-Human Primate (Cercopithecus campbelli) Auditory Laterality[END_REF].

A broad range of animal studies support the link between perceptual laterality and emotional state [START_REF] Rogers | Divided brains: The biology and behaviour of brain asymmetries[END_REF][START_REF] Versace | Forelimb preferences in human beings and other species: multiple models for testing hypotheses on lateralization[END_REF][START_REF] Vallortigara | Laterality at the neural, cognitive, and behavioral levels[END_REF]. The Valence-Specific Hypothesis (VSH) posits that the left side of the brain is specialised for processing positive emotions and the right side for processing negative emotions [START_REF] Ahern | Differential lateralization for positive versus negative emotion[END_REF][START_REF] Davidson | Hemispheric asymmetry and emotion[END_REF][START_REF] Wedding | Hemispheric differences in the perception of positive and negative faces[END_REF][START_REF] Hook-Costigan | Eye Preferences in Common Marmosets (Callithrix jacchus): Influence of age, stimulus, and hand preference[END_REF]. The predominance of the left visual field preference (i.e. suggesting a contralateral right hemisphere processing) varies with the level of negative emotions during social interactions and exposures to negative stimuli (nonhuman primates: Casperd & Dunbard, 1996;[START_REF] Baraud | Social laterality and 'transversality' in two species of mangabeys: influences of rank and implication for hemispheric specialization[END_REF][START_REF] Quaresmini | Social environment elicits lateralized behaviors in gorillas (Gorilla gorilla gorilla) and chimpanzees (Pan troglodytes)[END_REF]dogs: Quaranta et al., 2007;[START_REF] Siniscalchi | Seeing Left-or Right-asymmetric tail wagging produces different emotional responses in dogs[END_REF]horses: Larose et al., 2006;[START_REF] Boyer Des Roches | Laterality and emotions: Visual laterality in the domestic horse (Equus caballus) differs with objects' emotional value[END_REF][START_REF] Austin | Lateralization of agonistic and vigilance responses in Przewalski horses (Equus przewalskii)[END_REF]birds: Vallortigara et al., 1999;cetaceans: Chanvallon et al., 2017 andhoneybees: Rogers &[START_REF] Rogers | Complementary specializations of the left and right sides of the honeybee brain[END_REF].

During public performances, characteristics of the audience influence performers' emotions and behaviours [START_REF] Bode | A palmar sweat investigation of the effect of audience variation upon stage fright[END_REF][START_REF] Larsen | Gaze avoidance: Personality and social judgments of people who avoid direct face-to-face contact[END_REF]. For example, audience size, actors' emotions and lateralised positioning are inter-connected, and actors tend to use their left visual field (i.e. right hemisphere) in anxious and less preferred situations [START_REF] Lemasson | Audience size influences actors' anxiety and associated postures on stage[END_REF]. The spatial disposition of an audience in a theatre layout is associated with different rates of galvanic skin responses and a quadri-frontal disposition (with spectators on all possible sides) increases actors' emotional scores [START_REF] Lemasson | Influence of theatre hall layout on actors' and spectators' emotions[END_REF].

The pleasantness, responsiveness and interest of an audience are known to affect performers' internal state [START_REF] Macintyre | The effects of audience pleasantness, audience familiarity, and speaking contexts on public speaking anxiety and willingness to speak[END_REF][START_REF] Macintyre | The effects of audience interest, responsiveness, and evaluation on public speaking anxiety and related variables[END_REF] [START_REF] Bassett | The effects of positive and negative audience responses on the autonomic arousal of student speakers[END_REF][START_REF] Gardiner | A Synthesis of Experimental Studies of Speech Communication Feedback[END_REF][START_REF] Pertaub | An Experiment on Public Speaking Anxiety in Response to Three Different Types of Virtual Audience[END_REF]. In contrast, supportive audiences have a positive impact on speakers' stress [START_REF] Yokoyama | Influence of the role of a partner on anxiety due to a physically threatening situation[END_REF][START_REF] Taylor | Effects of a supportive or an unsupportive audience on biological and psychological responses to stress[END_REF]. We know that looking at angry/happy faces triggers more/less gaze avoidance especially when the observer is feeling anxious [START_REF] Roelofs | Gaze direction differentially affects avoidance tendencies to happy and angry faces in socially anxious individuals[END_REF]. However, to our knowledge, no study has evaluated the influence of an audience's attitude on performers' behavioural laterality.

We tested experimentally the impact of two contrasting controlled audience attitudes (Friendly/Hostile) on professional actors' spontaneous emotions and lateralised behaviour. Emotions were estimated by analysing their speech as stress is known to reduce speech fluency [START_REF] Buchanan | Acute stress reduces speech fluency[END_REF]. Lateralised behaviours were evaluated by head orientations and gaze directions. We hypothesised that left-side orientations and direct gazes would be fewer in the negative situation than in the positive situation.

METHODS

Since our study was only observational, with informed consents from participants, no further ethical authorisations were requested according to the French law. Following the recommendations of the European General Data Protection Regulation, this study was registered under the certificate #2-17049\UMR6552 by the Data Protection Service.

-Experimental setup

The study was conducted during two theatrical representations by the "Fabrique Autonome des Acteurs" (Moussey City, France) on August 30 th and the 31 st 2017. Ten professional actors, 6 women and 4 men, between 24 and 47 years old, took part in a play including ten monologues, lasting about 4 minutes each. During a given monologue, only one actor spoke and sat on a chair placed in front of the spectators. Each actor had the leading role in one monologue only while all the other actors remained on stage, sitting silently on chairs but down-stage.

All the monologues included a reference to animals and/or animal sciences. The ten monologues were delivered their lines consecutively and in the same order during the two representations. The actors and the spectators knew they were contributing to an ethological research project but were totally naive as to the scientific objectives, measurements and experimental variables. They were informed only that they had to remain sitting on the offered chair. Respectively 34 and 31 spectators attended the representations. The spectators differed between the two representations.

The two representations were identical so that the only experimental variable was the spectators' attitude. Just before the start of a representation, the spectators were informed that they had to contribute by being Friendly on

A u t h o r a c c e p t e d m a n u s c r i p t
the first day (i.e. to over express all their positive feelings) and Hostile on the second day (i.e. to express their positive feelings discreetly and to exaggerate all their negative feelings). Each leading actor was filmed using a Sony DCR-SR35 camera, positioned centrally, behind the spectators.

-Selection of texts and direction of actors

The texts for the ten monologues were chosen based on two criteria. First, all texts talked about animals and were mainly descriptive in order to be dramaturgically coherent. Second, all texts had to be emotionally "neutral" for the actors, such as naturalistic descriptions (i.e. three extracts of Darwin's "Voyage autour du monde à bord du Beagle", two extracts of Maëterlinck's "La vie des abeilles", one extract of Lorenz' "L'agression") and extracts of novels (i.e. three extracts of Mac Carthy's "La trilogie des plaines", one extract of London's "Construire un feu") in French. Prior work with the actors focused on some specific parts of the text (like active verbs) but never concerned the characters played.

-Data collection

The videos of all the entire monologues were scored using Boris software v.6.0.5 © 2012-2018. Using continuous focal sampling, we scored indicators of change in emotional state through the leading actor's speech: Mistakes (i.e. word omission, word addition, word error and word inversion, after comparing to the original text) and Silent pauses (total duration and number of occurrences) (Buchana et al., 2014;[START_REF] Lemasson | Audience size influences actors' anxiety and associated postures on stage[END_REF].

Durations were measured with a precision of 0.001s, and silences lasting more than 0.1s were considered a pause.

In addition, using instantaneous scan sampling, every 5 seconds, we measured (independently from one another) head orientations and gaze directions of the leading actor. Head orientations were categorised either as "Front" (i.e. facing the spectators), "Right" (i.e. right side oriented towards the spectators at an angle of more than 45°) or "Left" (i.e. left side oriented towards the spectators at an angle of more than 45°). Gazes were also categorised either as "Front" (i.e. directed towards the centre of the audience), the "Periphery" (i.e. directed towards the farthest sides of the audience) or "Undirected" (i.e. not directed towards the audience).

-Data analyses Given our small sample size (N = 10 actors), we only ran non-parametric statistical tests. Using Wilcoxon matched pair tests, we analysed the influence of the public's attitude on the actors' behaviours (speech, head orientation, gaze direction). We compared frequencies and durations for focal variables and percentages for scan variables. To run the data analysis, we used Rstudio software v. 1.0.146 © 2009-2016 (package: coin)

-Influence of the audience's attitude on the actors' speech

Speech analyses revealed that a Hostile public was associated with longer silence durations (V = 6 and p = 0.03) and a greater number of silences (Wilcoxon test: V = 5 and p = 0.04) than a Friendly public (Fig. 1). However, the public's attitude had no effect on the number of mistakes made by the actors during their monologues (Median number of mistakes for each representation = 3.5, Wilcoxon test: V = 17 and p = 0.67).

FIGURE 1 ABOUT HERE

-Influence of the audience's attitude on the actors' positions and gazes

We found that the public's attitude influenced orientation of actors' head. Indeed, actors' head was directed more to the Left when the public was Hostile than when it was Friendly (V = 6 and p = 0.027,

FIGURE 2 ABOUT HERE

These results also showed that gaze direction was influenced by the public's attitude. When the public was Friendly, actors gazed more towards the centre of the audience (V = 53 and p = 0.006, Fig. 3), and less towards the periphery of the audience (V = 0 and p = 0.002, Fig. 3) than when acting in front of a hostile audience. However, the audience's attitude did not influence the time spent with undirected gazes (V = 29 and p = 0.92).

FIGURE 3 ABOUT HERE

DISCUSSION

In line with our predictions, our data showed that actors presented spontaneously more often their lefthemiface to the public when the audience expressed a hostile rather than a friendly attitude. A hostile audience impaired actors' speech fluency. However, right-side orientations did not increase with a friendly public.

Nevertheless, the actors gazed more at the public when the audience was friendly but more to the periphery when it was hostile.

A u t h o r a c c e p t e d m a n u s c r i p t
Gaze avoidance is strongly related to emotional state and is a way for people to decrease their stress [START_REF] Larsen | Gaze avoidance: Personality and social judgments of people who avoid direct face-to-face contact[END_REF]. A vast majority of reports indicate that a higher percentage of eye contact between communicators is typically associated with more positive attitudes between the communicators [START_REF] Mehrabian | Significance of posture and position in the communication of attitude and status relationships[END_REF].

People make significantly more eye contacts with liked addressees than with disliked addressees [START_REF] Mehrabian | Significance of posture and position in the communication of attitude and status relationships[END_REF]. Perceiving direct/averted gazes activates the approach/avoidance motivational brain systems thus influencing the outcome of an interaction [START_REF] Hietanen | Seeing direct and averted gaze activates the approach-avoidance motivational brain systems[END_REF]. The lack of responsiveness and pleasantness of hostile observers certainly affected actors' emotional states and motivation to engage in long-lasting exchanges.

Binocular gazes by horses towards positive stimuli have been observed (de Boyer des Roches et al., 2018), suggesting flexibility in hemispheric specialisation for processing positive emotions.

The increase of left-hemiface (i.e. right brain hemisphere control) exposure in the hostile audience situation is consistent with the Valence Specific Hypothesis (e.g. [START_REF] Hook-Costigan | Eye Preferences in Common Marmosets (Callithrix jacchus): Influence of age, stimulus, and hand preference[END_REF]. Indeed, acting in front of a non-responsive audience certainly created an unpleasant atmosphere and thus a bias in favour of a right-hemisphere processing of negative emotions. Alternatively, this may have been a way actors spontaneously and unconsciously adapted to the situation by favouring the exposure of their left hemiface, which typically expresses emotions more intensively [START_REF] Asthana | Hemifacial asymmetry in emotion expressions[END_REF], possibly in order to increase the chances of eliciting more positive responses from the audience. Previous authors showed that, when photographers ask people to portray a variety of emotions, they present more often the left side of their face [START_REF] Nicholls | Laterality of expression in portraiture: putting your best cheek forward[END_REF]. Portraits of left side posers received higher emotional expressive rates [START_REF] Nicholls | The effect of left and right poses on the expression of facial emotion[END_REF]. Finally, actors may have been more surprised and hence more attentive towards the hostile public, and this may also be reflected by their longer silences that would trigger a right-hemisphere bias [START_REF] Lemasson | Influence of theatre hall layout on actors' and spectators' emotions[END_REF].

Although this study would deserve replication with larger sample sizes and repeated trials in order to make these conclusions more solid, it brings new findings that consolidate an earlier study showing that lefthemiface exposure of actors increased in more anxiety-provoking theatrical situations (i.e. larger public) [START_REF] Lemasson | Audience size influences actors' anxiety and associated postures on stage[END_REF]

  Fig 2). However, the public's attitude did not influence other head orientations (Front: V = 40 and p = 0.23; Right: V = 24 and p = 0.77; Fig 2).

Fig. 1

 1 Fig.1 Actors' silences in relation to the public's attitude (Friendly or Hostile). Left: Total duration; right: number of occurrences. Wilcoxon tests: * p < 0.05.

Fig. 2

 2 Fig.2 Head orientation to the public's attitude. Times (in percent) spent by the actor in each orientation. Wilcoxon tests: * p < 0.05.

Fig. 3 Fig. 1

 31 Fig.3 Eyes orientation in relation to the public's attitude. Times (in percent) spent by the actor in each orientation. Wilcoxon tests: ** p < 0.01.

Fig. 2

 2 Fig.2 Head orientation to the public's attitude. Times (in percent) spent by the actor in each orientation. Wilcoxon tests: * p < 0.05.

Fig. 3

 3 Fig.3 Eyes orientation in relation to the public's attitude. Times (in percent) spent by the actor in each orientation. Wilcoxon tests: ** p < 0.01.

  Emotions; Public performance; Audience effect; Laterality; Theatre.

	A u
	t
	h
	o r
	a
	c c e
	p
	t e
	d
	m
	a
	n u
	s c
	r i
	p t

  . Negative responses from an audience typically

	increase speakers' anxiety
	A u
	t
	h
	o r
	a
	c c e
	p
	t e
	d
	m
	a
	n u
	s c
	r i
	p t

  . It confirms the possibility to run social human laterality studies in naturalistic contexts.University and C.N.R.S., as well as by the "Fabrique Autonome des Acteurs" with the support of the Fondation Daniel & Nina Carasso and the French Ministry of Culture.

	FIGURE CAPTIONS
	A u A u A u A u A u A u
	t t t t t t
	h h h h h h
	o r o r o r o r o r o r
	a a a a a a
	c c e c c e c c e c c e c c e c c e
	p p p p p p
	t e t e t e t e t e t e
	d d d d d d
	m m m m m m
	a a a a a a
	n u n u n u n u n u n u
	s c s c s c s c s c s c
	r i r i r i r i r i r i
	p t p t p t p t p t p t

ACKNOWLEDGEMENTS

We greatly appreciate the friendly participation of all actors and spectators in this study. We are grateful to Antoine L'Azou for his help in coordinating the project. We also thank Corinne Jola for her comments on the protocol. Finally, we thank Ann Cloarec for checking the English text. This study was funded by Rennes 1


Estelle Rousselet

Alain Martelli

Mireille Chevallet

Hélène Diemer

Alain Van Dorsselaer

Thierry Rabilloud

Jean-Marc Moulis
email: jean-marc.moulis@cea.fr

Zinc adaptation and resistance to cadmium toxicity in mammalian cells. Molecular insight by proteomic analysis

Keywords: High Zinc Resistant HeLa cells, SERCA: sarco-endoplasmic reticulum Ca 2+ -ATPase, ICP-OES: Inductively coupled plasma optical emission spectroscopy, LC 50 : lethal concentration for 50% of the cells, HSP: Heat Shock Proteins, UPR: Unfolded Protein Response, Hop: Hsp70/Hsp90 organizing protein, HPPD : 4-hydroxyphenylpyruvate dioxygenase, NTBC 4-hydroxyphenylpyruvate, calcium, epithelial cells, 2D electrophoresis, tyrosine 2

Introduction

Zinc is an essential metal as a component of a wide variety of proteins, including transcription factors, metalloenzymes, and other proteins with various functions [START_REF] Berg | The galvanization of biology: a growing appreciation for the roles of zinc[END_REF]. However, large or improperly handled zinc concentrations in cells are toxic [START_REF] Vallee | The biochemical basis of zinc physiology[END_REF]. Reciprocally, too little zinc induces apoptosis [START_REF] Chimienti | Role of cellular zinc in programmed cell death: temporal relationship between zinc depletion, activation of caspases, and cleavage of Sp family transcription factors[END_REF] since zinc is an inhibitor of caspase 3 [START_REF] Truong-Tran | The role of zinc in caspase activation and apoptotic cell death[END_REF]. Consequently zinc homeostasis is precisely maintained [START_REF] Cousins | Mammalian zinc transport, trafficking, and signals[END_REF].

Zinc and cadmium share a range of physico-chemical properties, but it is not known whether their toxicity mechanisms are related or similar [START_REF] Martelli | Cadmium toxicity in animal cells by interference with essential metals[END_REF]. Cadmium is well recognized as an industrial pollutant and a food contaminant which accumulates with a half-life of more than 10 years in the body due to inefficient excretion mechanisms. Mercury is also a chemical sharing the same electronic configuration with filled d orbitals. However some properties of mercury, including very strong formation constants with various ligands, common two ligandcoordination, large volatility of the element, or ease of alkylation, trigger different toxicity and detoxification mechanisms in living cells and target different organs in mammals, such as the central nervous system, as compared to zinc and cadmium [START_REF] Rooney | The role of thiols, dithiols, nutritional factors and interacting ligands in the toxicology of mercury[END_REF]. Cadmium is deleterious to HeLa cells, but adaptation to the toxic metal has been demonstrated [START_REF] Cigliano | Analysis of metalregulated metallothionein and heat shock gene expression in HeLa-derived cadmium-resistant cells[END_REF]. A human epithelial cell line derived from HeLa cells by increased resistance to zinc, called HZR, has been previously developed [START_REF] Chimienti | Zinc resistance impairs sensitivity to oxidative stress in HeLa cells: protection through metallothioneins expression[END_REF] and it provides a convenient model to study mechanisms of resistance to heavy metals such as zinc and cadmium. This adapted cell line is maintained in 200 µM of zinc in the culture medium, a concentration that is more than 10 times the average zinc concentration (10-15 µM) found in human plasma [START_REF] Sullivan | Metallothionein expression is increased in monocytes and erythrocytes of young men during zinc supplementation[END_REF].

Once inside cells, cadmium leads to a diverse and complex series of events that may culminate with cell death. To attenuate cadmium toxicity, cells may respond with various strategies. These include proteins and activities that: (i) sequester cadmium, such as metallothioneins (MT), to prevent its interaction with sensitive cellular targets; (ii) increase glutathione synthesis and detoxify reactive oxygen species that are generated by cadmium;

(iii) repair damage to cellular components such as DNA; and (iv) help folding or degrade unfolded proteins [START_REF] Beyersmann | Cadmium, gene regulation, and cellular signalling in mammalian cells[END_REF]. In response to the endoplasmic reticulum (ER) stress that is induced by impairment of protein folding, cells activate a pathway known as the unfolded protein response (UPR) [START_REF] Rutkowski | A trip to the ER: coping with stress[END_REF]. The UPR is a complex mechanism that includes increased synthesis of heat shock proteins (HSP) acting as mediators of protein folding to maintain or recover their activity. A mechanism that contributes to maintain cellular homeostasis is the elimination of unfolded proteins through the energy-dependent, ubiquitin-proteasome degradation pathway [START_REF] Meusser | ERAD: the long road to destruction[END_REF]. This pathway involves two steps. First, the target protein is conjugated with ubiquitin molecules at lysine residues, then the ubiquitin-tagged substrate is transferred to the 26S proteasome, a multisubunit complex consisting of a 20S barrel-shaped proteolytic core and a 19S cap-like regulatory complex.

A candid approach to gain insight into the differences between zinc-resistant HZR and reference HeLa cells has been implemented by obtaining high resolution 2-DE of total cellular extracts. Comparison of the two proteomes revealed a short list of proteins that differed between the two cell lines: they pointed to some of the above mentioned biological functions previously shown to be perturbed by cadmium. These functions have been probed by use of specific inhibitors and no unique activity can be singled out to explain the resistant phenotype of HZR cells. Rather, detailed analysis of the suggested pathways indicates a likely important role for signaling pathways, as shown by the sensitivity of resistant cells to perturbed calcium homeostasis. In addition, these studies also highlight the importance of 4hydroxyphenylpyruvate dioxygenase, an enzyme of the tyrosine catabolic pathway, in the adaptation to zinc and the resistance against cadmium toxicity in HZR cells.

Materials and Methods

Cell Culture and Preparation of cell lysates

Epithelial human cervix carcinoma HeLa cells were grown in DMEM (Sigma-Aldrich, St Louis, MO, USA) with 2 mM L-glutamine and 5% heat-inactivated FBS at 37°C with a gas mixture of 5% CO 2 and 95% air. HZR cells [START_REF] Chimienti | Zinc resistance impairs sensitivity to oxidative stress in HeLa cells: protection through metallothioneins expression[END_REF] were routinely grown as HeLa cells in the same medium supplemented with 200 µM zinc sulfate. Exchange of zinc sulfate for zinc acetate did not change the behavior of HZR cells.

HeLa and HZR cells (40 x 10 6 ) were harvested by centrifugation, rinsed three times in 1 mL phosphate-buffered saline and pellets were suspended in homogenization buffer (0.25 M sucrose, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA). A buffer volume approximately equal to the packed cell volume was used. The suspension was transferred to a polyallomer ultracentrifuge tubes, and the cells were lysed by the addition of 4 volumes (respective to the suspension volume) of 8.75 M urea, 2.5 M thiourea, 25 mM spermine, and 50 mM dithiothreitol. After 1 hour at room temperature, the extracts were centrifuged (30 min at 200,000 x g). The supernatant was collected and the protein content was determined by the Bio-Rad protein assay using bovine serum albumin as a standard. A total of 500 µg of proteins were diluted in 1 mL rehydratation buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.4% ampholytes, 20 mM DTT). The protein extracts were stored at -20 °C.

2-DE

The data reported herein were obtained with samples prepared from at least three independent cultures of each cell line. For a given lysate, at least two gels were prepared, one to localize differentially produced spots between the two cell lines by silver staining and a second to confirm the first by visualizing with a mass spectrometry-compatible stain, and to extract proteins of interest.

The first dimension of electrophoresis was performed with immobilized pH gradients for isoelectric focusing. Non linear 4-8 and zoom 5.5-7.5 pH gradients were used. Homemade pH gradient plates were cast and cut into 4-mm-wide strips [START_REF] Gorg | Two-dimensional electrophoresis of proteins in an immobilized pH 4-12 gradient[END_REF][START_REF] Rabilloud | Sample application by in-gel rehydration improves the resolution of two-dimensional electrophoresis with immobilized pH gradients in the first dimension[END_REF]. The samples were applied onto the strips by in-gel rehydration overnight using a thiourea-urea mixture as denaturing agent [START_REF] Rabilloud | Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients[END_REF]. IEF was carried out for 60,000 Vh at a maximum of 3000 V using the Multiphor II system (Amersham-Pharmacia, Sweden). Strips were then equilibrated for 20 min first in 0.15 M BisTris/0.1 M HCl, 6 M urea, 2.5% SDS, 30% glycerol, 0.5 M DTT and then in 0.15 M BisTris/0.1 M HCl, 6 M urea, 2.5% SDS, 30% glycerol, 0.3 M iodoacetamide. Strips were placed on top of a SDS-polyacrylamide gel. After migration, the gels were stained either with silver [START_REF] Sinha | A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of proteins after two-dimensional electrophoresis[END_REF], or with colloidal Coomassie Blue when protein identification was sought [START_REF] Neuhoff | Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250[END_REF]. Expression ratios were estimated by image analysis of pairs of gels with the Delta2D software (DECODON GmbH, Greifswald, Germany). At least three pairs (HeLa vs HZR cells) of gels run in parallel were analyzed and statistics reported in Table 1 are for between 3 and 6 measurements.

In Gel Digestion and MALDI-TOF-MS Analysis

Excising gel slices, rinsing, and reduction/alkylation steps were performed on a MassPREP station (Micromass, Manchester, UK) as described previously [START_REF] Wagner | A method for detection of overoxidation of cysteines: peroxiredoxins are oxidized in vivo at the active-site cysteine during oxidative stress[END_REF]. Gel pieces were completely vacuum-dried before digestion. Three volumes of freshly diluted 12.5 ng/µl trypsin (Promega, Madison, WI) in 25 mM NH 4 HCO 3 were added to the volume of the dried gel. Digestion was performed at 35°C overnight. Then, the samples were again vacuum-dried for 5 min and 5 µl of 35% H 2 O:60% acetonitrile:5% HCOOH were added to extract peptides.

The mixture was sonicated for 5 min and centrifuged for 5 min. The supernatant was recovered and the procedure was repeated once with the pellet.

Mass measurements were carried out on an Ultraflex TM MALDI-TOF/TOF spectrometer (Bruker-Daltonik GmbH, Bremen, Germany). The instrument was used at a maximum accelerating potential of 20 kV and was operated in reflector-positive mode. Sample preparation was performed with the dried droplet method using a mixture of 0.5 µl of sample with 0.5 µl of matrix solution. The matrix solution was prepared from a saturated solution of α-cyano-4-hydroxycinnamic acid in H 2 O:50% acetonitrile diluted 3 times in water. Internal calibration was performed with tryptic peptides resulting from autodigestion of trypsin (monoisotopic masses at m/z = 842.5; m/z = 1045.6; and m/z = 2211.1).

Monoisotopic peptide masses were assigned and used for databases searches using the search engine MASCOT (Matrix Science, London, UK) [START_REF] Perkins | Probability-based protein identification by searching sequence databases using mass spectrometry data[END_REF]. All human proteins present in Swiss-Prot were used without any pI and M r restrictions. The peptide mass error was limited to 70 ppm, one possible missed cleavage was accepted.

MTT cell viability and other assays

The cytotoxicity of cadmium acetate, in addition to different drugs, on HeLa and HZR cells was determined after 24 hours of exposure by a modified (3-[4, 5-dimethylthiazol-2-yl]-2, 5diphenyl tetrazolium) bromide (MTT) method [START_REF] Carmichael | Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing[END_REF]. Three thousand cells in 100 µl of growth medium were plated in 96-well plates and incubated for 24 hours before probing with a mix of cadmium acetate and different drugs. For HZR cells, 200 µM of zinc used to maintain these cells were replaced by cadmium. MTT, lactacystin, thapsigargin, and tunicamycin were obtained from Sigma Chemical Co (S t Louis, MO, USA). NTBC was a kind gift of Dr David King (Swedish Orphan International AB, Stockholm, Sweden): stock solutions were prepared in ethanol and diluted in the medium to a maximum of 0.1% ethanol. Viability was measured 24 hours later: ten μl of 5 mg/ml MTT were added in each well and the cells were incubated at 37°C for 3 hours. The medium was removed, and formazan was dissolved in 100 µl of DMSO. The optical density was measured at 560 nm with a microplate reader (Multiskan,

Labsystem RC). The percentage of viable cells (%) was calculated as [(A-B)/(C-B)] × 100

where A = OD 560 of the treated sample, B = OD 560 of the background absorbance, and C = OD 560 of the reference cells not exposed to the chemical compound being tested. Background absorbance was estimated by lysing a row of reference cells with 1% Triton X-100 before applying MTT. Error bars were calculated from 8 measurements in several separate experiments. The data were fitted to a sigmoid curve from which LC 50 values were derived as the 50% intersecting points. The statistical significance between data sets was assessed in Microsoft Excel by first comparing variances with a Fischer-Snedecor (F-) test for unpaired datasets at the 0.05 probability point. Student t-tests were then performed according to the outcome for F that generally indicated unequal variances.

For intracellular metal determinations, cells were harvested by trypsination and washed twice with PBS without calcium and magnesium and once with the same buffer containing 50 mM EDTA. Pellets were vacuum-dried and mineralized in 70% nitric acid before analysis with Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) with a Varian, Vista MPX instrument [START_REF] Martelli | Folding and turnover of human iron regulatory protein 1 depend on its subcellular localization[END_REF]. The metal content was referred to the amount of cells in the analyzed pellet.

Homogentisate dioxygenase was measured spectrophotometrically following formation of maleylacetoacetate at 330 nm, using an extinction coefficient of 13500 M -1 cm -1 [START_REF] Schmidt | Murine liver homogentisate 1,2-dioxygenase. Purification to homogeneity and novel biochemical properties[END_REF]. First cell extracts were incubated anaerobically with 100 µM ferrous sulfate and 200 µM ascorbate for 10 min to reactivate any enzyme that may have been iron-depleted upon breaking the cells. The assay was carried out aerobically in 20 mM Mes, 80 mM NaCl at pH 6.2 with 2 mM homogentisate and it was started by adding the enzyme.

The intracellular melanin concentration was measured by a previously devised method [START_REF] Siegrist | In situ melanin assay for MSH using mouse B16 melanoma cells in culture[END_REF] with ca. 5x10 6 cell-samples and a reference plot obtained with synthetic melanin prepared by oxidation of tyrosine with hydrogen peroxide (Sigma, reference M8631) and dissolved in 0.85 N KOH. Briefly, cells were washed with 0.02% EDTA and lysed with 0.5 ml water followed by two cycles of freezing and thawing. After centrifugation, pellets were washed three times with 5% trichloroacetic acid, twice with a cold mixture of ethanol/ethyl ether (3/1) and once with cold ether. The air-dried pellets were dissolved in 0.3 ml of 0.85 N KOH by heating to 100°C for 10 min. The absorbance was measured at 400 nm.

Quantitative RT-PCR

RNA extractions were carried out with the total Quick RNA TM kit (Talent s.r.l., Trieste, Italy).

Results

Comparative Proteome analysis of HeLa and HZR cells.

To probe the mechanisms responsible for zinc resistance of HZR cells, 2-DE has been implemented to compare the HZR proteome with that of parental HeLa cells (Figure 1).

Lysates of four different cultures of each cell line were analyzed. The general pattern displayed by all gels is quite similar, despite the addition of 200 µM zinc to the medium used to grow HZR cells (Figure 1). This indicates that such gels are highly reproducible and that only few proteins have changed intensities or positions between the two cell lines. Therefore these differences are probably not due to a general and massive effect of zinc overload in HZR cells, and they are likely to give insight into the mechanism responsible for the phenotype of HZR cells. More detailed data were obtained with pI gradients between 5.5 and 7.5. Figure 2 shows silver-staining of representative gels obtained after separation of 500 µg protein samples from HeLa and HZR cells, respectively. Spots of different intensities or absent in one of the two gels were excised and subjected to trypsin digestion, MALDI-TOF mass spectra measurements, and database searching. Table 1 summarizes the proteins, the concentrations of which change between HeLa and HZR cells. This list includes a cochaperone, stress induced phosphoprotein-1 or Hsp70/Hsp90 organizing protein (Hop), enzymes or subunits involved in oxidoreductase activities, thioredoxin reductase, flavoprotein subunit of succinate dehydrogenase, 4-hydroxyphenyl-pyruvate dioxygenase, and carbonic anhydrase II.

As Chimienti et al. [START_REF] Chimienti | Zinc resistance impairs sensitivity to oxidative stress in HeLa cells: protection through metallothioneins expression[END_REF] previously pointed out, metallothioneins largely contribute to zinc adaptation of HZR cells. In an attempt to visualize metallothionein up-regulation by 2-DE, higher concentration gels were applied in the second dimension. Two spots of apparent 6.5 kDa mass were detected. The protein labeled M1 is of higher intensity in gels of HZR extracts than in those of HeLa cells (Figure 3) but its pI is lower than expected for metallothionein. Indeed, identification with mass spectrometry revealed that M1 did not correspond to metallothionein but to ubiquitin. M2 is the ubiquitin-like molecule NEDD8, the function of which includes regulation of the cell cycle, but its intensity is weak and it does not vary between the two cell lines under study (Figure 3).

The role of the proteins listed in Table 1 in the cellular response to divalent metal stress has been probed in the following.

ER stress and sensitivity to cadmium toxicity

Stress-induced phosphoprotein 1 (Hop) is a 60-kDa co-chaperone that mediates the association of the heat shock proteins Hsp70 and Hsp90 [START_REF] Chen | Hop as an adaptor in the heat shock protein 70 (Hsp70) and hsp90 chaperone machinery[END_REF]. The function of Hop has been best characterized in in vitro systems examining the assembly of the progesterone receptor [START_REF] Kosano | The assembly of progesterone receptor-hsp90 complexes using purified proteins[END_REF] and glucocorticoid receptor [START_REF] Dittmar | Folding of the glucocorticoid receptor by the reconstituted Hsp90-based chaperone machinery. The initial hsp90.p60.hsp70-dependent step is sufficient for creating the steroid binding conformation[END_REF] into hormone-binding competent hetero-complexes [START_REF] Kimmins | Maturation of steroid receptors: an example of functional cooperation among molecular chaperones and their associated proteins[END_REF].

In previous investigations, cadmium has been reported to interfere with protein folding, leading to accumulation of misfolded proteins in ER [START_REF] Liu | Cadmium induces the expression of Grp78, an endoplasmic reticulum molecular chaperone, in LLC-PK1 renal epithelial cells[END_REF], and HZR cells have been shown to sustain cadmium concentrations almost as high as zinc ones over 24 hours without increased lethality (Rousselet et al. in preparation). To estimate the endoplasmic reticulum stress that cells studied herein may experience under cadmium exposure, two different drugs were applied in addition to the toxic metal. The sesquiterpene lactone, thapsigargin, inhibits sarcoendoplasmic reticulum Ca 2+ -ATPase (SERCA) and consequently depletes the intracellular calcium stores [START_REF] Inesi | Studies of Ca 2+ ATPase (SERCA) inhibition[END_REF] and tunicamycin inhibits N-acetylglucosamine transferases, so preventing glycosylation of newly synthesized glycoproteins.

Viability tests were performed with the MTT assay in the presence of cadmium acetate for HeLa and HZR cells with 50 nM of thapsigargin or tunicamycin. These concentrations of inhibitors were chosen because they minimally decrease viability to the same extent (less than ca. 20%) for both cell lines. After 24 hours of exposure, the half-maximal lethal concentrations were about 7 µM of cadmium for HeLa cells exposed to 50 nM thapsigargin or 50 nM tunicamycin (Figure 4A). These values are not significantly different than those measured in the absence of thapsigargin or tunicamycin (Rousselet et al. in preparation), indicating that these concentrations of ER stressors do not sensitize HeLa cells to the cadmium insult.

In the case of HZR cells, replacement of the 200 µM zinc sulfate by varying cadmium concentrations for 24 hours and using the same concentration of thapsigargin as for HeLa cells indicated that these cells were far more sensitive to cadmium than without thapsigargin (Figure 4B). Cadmium half-maximal lethal dose measured after 24 hours of exposure was 25 µM with 50 nM thapsigargin. This value is ten times smaller than in the absence of thapsigargin. Exposure of HZR cells to 50 nM tunicamycin (Figure 4B) led to a cadmium half-maximal lethal dose of 165 µM, but this increased sensitivity to cadmium with tunicamycin is not as large as with thapsigargin. Therefore, inhibition of SERCA by thapsigargin and subsequent disruption of calcium homeostasis appears as a more potent stress that enhances cadmium toxicity than inhibition of glycosylation. These experiments indicate that increasing ER stress decreases cadmium resistance toward cadmium, in a very sensitive way for HZR cells treated with thapsigargin.

Since HZR cells showed a strong sensitivity to the presence of 50 nM thapsigargin, the metal content of cells under the conditions implemented to assess viability in the presence of cadmium was measured by ICP-OES. Thapsigargin lowered cadmium accumulation in HeLa cells, but not in HZR cells, at least in the cadmium concentration range that remained accessible with 50 nM of the drug. It follows that the loss of viability of HZR cells is not directly linked to intra-cellular cadmium in the presence of thapsigargin.

Proteasomal degradation and sensitivity to cadmium toxicity

As shown in Figure 3, ubiquitin was found to be up-regulated in HZR cells maintained in 200 µM zinc. Although ubiquitin fulfills widespread cellular functions [START_REF] Herrmann | Ubiquitin and ubiquitin-like proteins in protein regulation[END_REF], labeling of misfolded proteins for removal by the proteasome is one of the major role of this small protein [START_REF] Meusser | ERAD: the long road to destruction[END_REF]. Upregulation of ubiquitin in HZR cells under standard conditions, i.e. with 200 µM zinc, may reveal enhanced protein turnover through the proteasome pathway. This pathway was probed by viability tests implementing the MTT assay on HeLa and HZR cells with increasing concentrations of lactacystin [START_REF] Fenteany | Lactacystin, proteasome function, and cell fate[END_REF] for 24 hours (Figure 5A). A proteasomal inhibitor concentration (2.5 µM) leaving about 80% of the cells under study viable after 24 hours of exposure was chosen in the following experiments. Half-maximal lethal concentration after 24 hour-exposure to cadmium and 2.5 µM lactacystin was 3 µM for HeLa cells (Figure 5B), as compared to 7 µM for cells exposed to cadmium alone (p value <0.001). In the case of HZR cells, the cadmium half-maximal lethal dose was 75 µM after 24 hours of exposure to 2.5 µM lactacystine (Figure 5C). As in the case of HeLa cells, this value is significantly smaller (p value <0.001) than that measured in the absence of lactacystin.

Qualitatively similar results were obtained by replacing the proteasomal irreversible inhibitor lactacystin by the reversible inhibitor MG132. These data highlight the involvement of the ubiquitin-proteasome pathway in the cellular response against cadmium toxicity in both HeLa and HZR cells.

Involvement of 4-hydroxyphenylpyruvate dioxygenase activity in zinc adaptation and cadmium toxicity for HZR cells

The 2-DE data revealed up-regulation of 4-hydroxyphenylpyruvate dioxygenase (HPPD) in HZR cells as compared to HeLa cells (Figures 1 and2). We have thus evaluated expression of the HPPD gene in both cell lines: relative levels of HPPD transcripts were measured by RT-PCR under standard growth conditions and compared to three different housekeeping genes.

The expression of the HPPD gene was found to be about five times higher in HZR than in HeLa cells (Figure 6). This value agrees with the concentration ratio estimated for the corresponding proteins (Table 1). Therefore HPPD overproduction in HZR cells occurs mainly by increased transcription or mRNA stabilization as compared to HeLa cells. The increased concentration of HPPD in HZR cells does not seem to be triggered by the high zinc concentration in which these cells are maintained. Indeed, HeLa cells exposed to zinc before analysis by 2-DE did not display higher HPPD concentrations than HeLa cells kept in a medium with a normal zinc concentration. However, the reversibility of the HZR phenotype [START_REF] Truong-Tran | The role of zinc in caspase activation and apoptotic cell death[END_REF] was mirrored by the decrease of HPPD in HZR cells kept without excess of zinc for 3 days. Therefore, increased HPPD is associated with the HZR phenotype characterized by resistance to both high zinc and cadmium concentrations, rather than with up-regulation induced by short-term exposure to zinc.

In order to know if the HPPD activity contributes to cadmium management in the presently studied cells, a specific inhibitor of the enzyme, 2-nitro-4-(trifluoromethyl) benzoyl-1,3cyclohexanedione (NTBC), was used [START_REF] Kavana | Interaction of (4-hydroxyphenyl)pyruvate dioxygenase with the specific inhibitor 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione[END_REF][START_REF] Lindstedt | Treatment of hereditary tyrosinaemia type I by inhibition of 4-hydroxyphenylpyruvate dioxygenase[END_REF]. HeLa and HZR cells were treated with different concentrations of NTBC for 24 hours. Up to 300 µM of NTBC, viability slowly decreased in a similar dose-dependent way for both cell lines (Figure 7A). The NTBC concentration of 5 µM minimally affected viability. It was added with cadmium acetate for 24 hours to measure the effect of the inhibitor on the cellular sensitivity to cadmium. For HeLa cells (Figure 7B), the half-maximal lethal cadmium concentration was unchanged in the presence of NTBC as compared to HeLa cells without NTBC (p value between data sets with and without the inhibitor= 0.63), whereas LC 50 was 90 µM of cadmium in the presence of 5 µM NTBC for HZR cells (Figure 7C). No significant changes of the intra-cellular concentrations of cadmium were measured for cells exposed to the same extra-cellular cadmium concentration, whether 5 µM of NTBC were added or not. Therefore HeLa cells did not seem to be more sensitive to cadmium in the presence of NTBC, but viability of HZR cells decreased by about three fold in these conditions (Figure 7C). The LC 50 shifts for HZR cells in the presence of NTBC varied with the concentration of the inhibitor, thus showing that NTBC sensitizes HZR cells for cadmium insult in a dose-dependent way.

Sensitivity of HZR cells to combined HPPD inhibition and cadmium treatments after long-term zinc removal

The above experiments were carried out by cadmium substitution of zinc in the HZR growth medium for 24 hours. However the zinc and cadmium resistance phenotype of HZR cells is reversible upon zinc withdrawal [START_REF] Chimienti | Zinc resistance impairs sensitivity to oxidative stress in HeLa cells: protection through metallothioneins expression[END_REF]. When zinc is removed from the growth medium for one week, not only the resistance against zinc decreases [START_REF] Chimienti | Zinc resistance impairs sensitivity to oxidative stress in HeLa cells: protection through metallothioneins expression[END_REF], but also the resistance against cadmium collapses to reach a LC 50 of 40 µM for 24 hours of exposure to cadmium (Rousselet et al. in preparation). These long-term zinc-depleted HZR cells were also treated with 5 µM or 10 µM NTBC in addition to cadmium for 24 hours. Viabilities obtained were similar for both NTBC concentrations giving LC 50 values of 40 µM on average. Therefore resistance against cadmium toxicity of long-term zinc-depleted HZR cells was greatly decreased as compared to zinc-maintained HZR cells, and NTBC no longer had any effect on the half-maximal lethal cadmium concentration for these cells, as was observed for HeLa cells (Figure 7B). These experiments demonstrate that cadmium handling by HZR cells depends on HPPD activity.

Cadmium sensitivity increases when this activity is inhibited (Figure 7) and phenotypic reversal by long-term zinc removal makes these cells insensitive to HPPD inhibition.

Combined effects of added tyrosine and HPPD inhibition on cadmium toxicity

The tyrosine degradation product, 4-hydroxyphenylpyruvate (HPP), is converted by HPPD to homogentisate [START_REF] Neve | Tissue distribution, intracellular localization and proteolytic processing of rat 4-hydroxyphenylpyruvate dioxygenase[END_REF]. Since HZR cells over-produce HPPD, tyrosine catabolism may be more active in these cells than in HeLa cells. Addition of tyrosine for 24 hours is not toxic for both cell lines up to 6 mM (not shown). The growth medium of HeLa and HZR cells, which already contains about 230 µM of this amino acid, was supplemented with sub-lethal 3 mM of L-tyrosine. Viability curves were recorded for HeLa and HZR cells under these conditions with increasing cadmium concentrations. They show a decrease of LC 50 with 3 mM Ltyrosine, which goes down to 5 µM for HeLa cells (Figure 8A) and 145 µM for HZR cells (Figure 8B), instead of 7 µM and 250 µM, respectively, for cells exposed to cadmium alone (Figure 8). Therefore, the burden of cadmium challenge is increased by the presence of sublethal tyrosine concentrations for both cell lines. The combined effects of NTBC and tyrosine on the sensitivity toward increasing cadmium concentration were also determined. The cadmium LC 50 obtained for HeLa cells in the presence of 3 mM tyrosine and 5 or 10 µM NTBC did not change as compared to tyrosine alone. In the case of HZR cells, the same experiments with both NTBC and tyrosine gave the same outcome, namely no significant changes of LC 50 for cadmium as compared to cells exposed to tyrosine alone.

The enzyme following HPPD in the tyrosine degradation pathway is homogentisate dioxygenase (HGO), that is mainly responsible for homogentisate withdrawal in mammalian cells. The activity of this enzyme in extracts of both HeLa and HZR cells has been measured.

As found for HPPD, HGO activity is ca. 5-fold higher in HZR cells (27 (SD 6) µM.min -1 .(mg of proteins) -1 ), as compared to HeLa cells (5.5 (SD 3.6) µM.min -1 .(mg of proteins) -1 ).

Therefore, 4-hydroxyphenylpyruvate conversion and ring opening are both enhanced in HZR cells as a result of adaptation to zinc.

Tyrosine has different biological roles in mammalian cells, as one of the component of proteins, but also as the precursor of catecholamines, such as dopamine, thyroid hormones, and melanin. The latter pigment has been shown to bind metals [START_REF] Szpoganicz | Metal binding by melanins: studies of colloidal dihydroxyindole-melanin, and its complexation by Cu(II) and Zn(II) ions[END_REF][START_REF] Kokkinou | The pigmentation of human iris influences the uptake and storing of zinc[END_REF], including zinc and cadmium, and it has been repeatedly proposed to participate in heavy metal detoxification [START_REF] Nicolaus | A critical review of the function of neuromelanin and an attempt to provide a unified theory[END_REF][START_REF] Loumbourdis | Impact of cadmium on liver pigmentary system of the frog Rana ridibunda[END_REF]. Homogentisate is a precursor of secreted melanin pigments (pyomelanin) in some microorganisms [START_REF] Plonka | Melanin synthesis in microorganisms--biotechnological and medical aspects[END_REF] and of plasma melanins in humans in cases of alkaptonuria with inflammation and darkening of connective tissues in particular [START_REF] Hegedus | Homogentisic acid and structurally related compounds as intermediates in plasma soluble melanin formation and in tissue toxicities[END_REF]. It is unknown at present if homogentisate, or a derivative, has the ability to produce heavy metal-protecting scavengers in mammalian cells. However, it is unlikely that these scavengers are in the form of intracellular melanin, since the measured amount in HZR cells (range 21-110 ng/µg of total proteins, 4 measurements) is not larger than in HeLa cells (range 45-180 ng/µg of total proteins, 3 measurements). Therefore the increased activities of HPPD and HGO in HZR cells do not favor melanin biosynthesis.

Discussion

In the present study adaptation to zinc overload developed by the HZR clone [START_REF] Chimienti | Zinc resistance impairs sensitivity to oxidative stress in HeLa cells: protection through metallothioneins expression[END_REF] was probed at the proteomic level. A list of proteins was found to be up-regulated in these cells, with the exception of carbonic anhydrase II that was decreased by a factor of 3 (Table 1). The corresponding gene was shown to be up-regulated by zinc deficiency in rat esophageal epithelia [START_REF] Liu | Modulation of gene expression in precancerous rat esophagus by dietary zinc deficit and replenishment[END_REF], in line with down-regulation upon zinc overload as noticed here. The biological pathways and functions suggested by comparative studies between the HZR clone and parental HeLa cells have been interrogated by use of specific inhibitors. Consequences on the sensitivity to cadmium have been measured to gauge the involvement of these pathways in the toxicity mechanism(s).

ER as a target of cadmium action in mammalian cells has been largely documented [START_REF] Hiramatsu | Rapid, transient induction of ER stress in the liver and kidney after acute exposure to heavy metal: evidence from transgenic sensor mice[END_REF][START_REF] Biagioli | Endoplasmic reticulum stress and alteration in calcium homeostasis are involved in cadmium-induced apoptosis[END_REF]. The viability of HZR cells was far more sensitive than that of HeLa cells to the simultaneous addition of the ER stressors tunicamycin or thapsigargin with cadmium. This strongly suggests that the HZR cells, able to accommodate high intracellular zinc concentrations [START_REF] Chimienti | Zinc resistance impairs sensitivity to oxidative stress in HeLa cells: protection through metallothioneins expression[END_REF], more heavily rely on ER functions than HeLa cells. However, ERassociated chaperones, such as Grp94, PDI, and Grp78, or other components of the quality control system of protein folding, such as Hsp90, do not show significant changes between the two cell lines (Figure 1). It means that HZR cells do not display signs of generalized misfolding of proteins as compared to HeLa cells before application of cadmium.

Once cadmium is added to the cultures, perturbed protein biosynthesis does occur as witnessed by the impairment of resistance against the toxic metal by small amounts of proteasomal inhibitors. But, under these conditions, HeLa cells appear as sensitive as HZR cells, since the decrease of viability is similar for the same inhibitor concentration. The applied cadmium concentrations are quite different between the two cell lines, but it has been found that viability and the intracellular concentrations of the metal are similarly related for both HeLa and HZR cells (Rousselet et al. in preparation). Therefore, the increased amount of ubiquitin in HZR cells does not seem to be needed for a more efficient degradation of misfolded proteins via the proteasome pathway. It may rather be due to some other role of ubiquitin, such as protein intracellular trafficking and other important biological functions [START_REF] Hurley | Ubiquitin-binding domains[END_REF].

The only detected exception to the similar concentration of chaperones between the two cell lines (Table 1) is Hop that is more abundant in HZR cells than in HeLa cells. Hop belongs to the large group of co-chaperones which regulates the activity of heat shock proteins through its tetratricopeptide repeat TPR1 and TPR2A domains [START_REF] Scheufler | Structure of TPR domainpeptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine[END_REF]. It participates in the maturation of the glucocorticoid receptor [START_REF] Carrigan | Functional comparison of human and Drosophila Hop reveals novel role in steroid receptor maturation[END_REF] and up-regulation of Hop has been demonstrated in activated macrophages [START_REF] Heine | Bacterial lipopolysaccharide induces expression of the stress response genes hop and H411[END_REF].

This single example among the several chaperones identified in 2-DE strongly suggests that adaptation of HZR cells to zinc is a well-targeted phenomenon involving only a small number of cellular components. Further experimental support for this statement is provided by the more acute sensitivity of these cells to the combined effects of cadmium and thapsigargin as compared to cadmium and tunicamycin. These two drugs target distinct activities, but they eventually trigger the same cellular Unfolded Protein Response. The larger effect of thapsigargin than that of tunicamycin in the presence of cadmium indicates that toxicity of the metal in HZR cells is more sensitive to adequate calcium intracellular distribution than to proper processing of membrane and secreted proteins.

Besides Hop, another protein being unambiguously present at higher concentration in HZR cells than in HeLa cells is the enzyme HPPD. It is an iron-and 2-oxo-glutarate-dependent enzyme that catalyzes the complex conversion, including hydroxylation, decarboxylation and rearrangement steps, of 4-hydroxyphenylpyruvate, the product of tyrosine aminotransferase, to homogentisate [START_REF] Purpero | Catalytic, noncatalytic, and inhibitory phenomena: kinetic analysis of (4-hydroxyphenyl)pyruvate dioxygenase from Arabidopsis thaliana[END_REF][START_REF] Nakai | Studies on a possible reaction intermediate of phydroxyphenylpyruvate dioxygenase[END_REF].

Missense mutations in the HPPD locus can cause two distinct genetic diseases, hereditary type III Tyrosinemia and Hawkinsinuria [START_REF] Tomoeda | Mutations in the 4hydroxyphenylpyruvic acid dioxygenase gene are responsible for tyrosinemia type III and hawkinsinuria[END_REF]. Type I Tyrosinemia is due to fumarylacetoacetate hydrolase deficiency [START_REF] Tanguay | Different molecular basis for fumarylacetoacetate hydrolase deficiency in the two clinical forms of hereditary tyrosinemia (type I)[END_REF], and, because symptoms of type III Tyrosinemia and Hawkinsinuria [START_REF] Scott | The genetic tyrosinemias[END_REF] are less severe than those of type I Tyrosinemia, the latter is treated by inhibition of HPPD with the 1,3 diketone NTBC [START_REF] Lindstedt | Treatment of hereditary tyrosinaemia type I by inhibition of 4-hydroxyphenylpyruvate dioxygenase[END_REF], hence avoiding the buildup of the deleterious metabolites fumarylacetoacetate and succinylacetone. These metabolic and clinical data clearly indicate that defaults of tyrosine catabolism strongly impact cellular fate.

HPPD is subjected to several post-translational processing events [START_REF] Neve | Tissue distribution, intracellular localization and proteolytic processing of rat 4-hydroxyphenylpyruvate dioxygenase[END_REF][START_REF] Aarenstrup | Expression and posttranslational modification of human 4-hydroxy-phenylpyruvate dioxygenase[END_REF], which may explain the presence of at least two spots on 2D gels assigned to HPPD (Figure 2, Table 1), but no variations between the relative intensities of these spots have been observed in our experiments. The HPPD transcript is more abundant in the HZR than in the parental HeLa cell line (Figure 6). Promoter analysis evidenced binding sites for several transcription factors, including Sp1, CREB, and C/EBP [START_REF] Aarenstrup | Expression and posttranslational modification of human 4-hydroxy-phenylpyruvate dioxygenase[END_REF]. The HPPD activity appeared more important in HZR cells than in HeLa cells, since its inhibition selectively sensitized the former cells to cadmium.

However there is no association between HPPD activity and cadmium traffic since NTBC did not change the amount of metal accumulated by cells. A modest impact of increased tyrosine on cadmium toxicity was measured. If tyrosine removal has a role in cadmium detoxification, inactivation of HPPD should enhance cadmium toxicity in the presence of large amounts of tyrosine. Instead, no additive effects of tyrosine and the HPPD inhibitor NTBC on the viability curves with cadmium were detected. The NTBC dose-dependent decrease of HZR cells viability in the presence of cadmium is thus not due to decreased tyrosine disposal.

The HPPD substrate, HPP, is the first compound produced in the tyrosine degradation pathway. It has also been shown to be a substrate of phenylpyruvate tautomerase, which produces the enol-form of HPP and is the same protein as macrophage migration inhibitory factor (MIF) [START_REF] Rosengren | The macrophage migration inhibitory factor MIF is a phenylpyruvate tautomerase[END_REF]. Whether increased depletion of HPP, as expected in HZR cells, influences the cytokine, including growth-promoting [START_REF] Mitchell | Tumor growth-promoting properties of macrophage migration inhibitory factor (MIF)[END_REF], function of MIF is unknown. Indeed, regulation of the balance between the two activities of this moonlighting protein is not elucidated, and the physiological meaning of the enzymatic reaction has been questioned [START_REF] Swope | Direct link between cytokine activity and a catalytic site for macrophage migration inhibitory factor[END_REF].

Yet, from what is known about regulation of enzymes involved in tyrosine turnover, including HPPD [START_REF] Aarenstrup | Expression and posttranslational modification of human 4-hydroxy-phenylpyruvate dioxygenase[END_REF], it is beyond doubt that signaling pathways regulate flow between the different metabolic routes available for tyrosine. In this respect, disruption of calcium homeostasis by thapsigargin in HZR cells, and its consequences on signaling pathways, strongly impacts the resistance of these cells against cadmium (Figure 4B). MIF may also be involved in regulating signaling pathways [START_REF] Aeberli | Endogenous macrophage migration inhibitory factor modulates glucocorticoid sensitivity in macrophages via effects on MAP kinase phosphatase-1 and p38 MAP kinase[END_REF].

The proteomic approach described herein and complementary experiments have revealed an unsuspected association between tyrosine catabolism and adaptation to zinc overload that also contributes to the toxicity of cadmium in mammalian cells. Very recently [START_REF] Collin | Vitamin E is essential for the tolerance of Arabidopsis thaliana to metal-induced oxidative stress[END_REF], upregulation of HPPD has been observed in Arabidopsis thaliana upon exposure to cadmium, as a means to increase vitamin E synthesis for which homogentisate is a precursor in plants.

Since this pathway is not present in animals, the association between HPPD activity and cadmium toxicity in different organisms will be worth investigating further.

Table 1. Identification of proteins differently present in HeLa and HZR cells a .

a The labeled spots in Figure 2 are indicated with their identification using MALDI-TOF with sequence coverage (% R). Swiss-Prot accession numbers, monomer masses and isoelectrofocusing points obtained for each protein are also given. The average expression ratio between HZR and HeLa cells estimated with the Delta2D software are indicated under E with standard errors in brackets as described in Materials and Methods.

b the protein was not detected in HeLa cells c the gels such as that in Figure 3 were not analyzed with the Delta2D software. micropreparative gels. Labeled spots were identified as reported in Table 1.

  Total RNA (1.4 µg) was reverse transcribed by the RevertAid TM H Minus M-MuLV Reverse Transcriptase (MBI Fermentas, Vilnius, Lithuania) at 42°C for one hour using a poly-T primer. Quantitative RT-PCR was performed with a Stratagene Mx3005P instrument as follows: the reaction mixture consisted of 12.5 µl of Full Velocity SYBR Green QPCR Master Mix (Stratagene, La Jolla, CA, USA), 140 nM of HPPD primers and 5 µl of 12.5 ng cDNA adjusted to 25 µl with water. Initial denaturation was at 95°C for 5 min, followed by amplification for 40 PCR-cycles at 95°C for 10 s and at 60°C for 30 s successively. PCR fluorescent signals were normalized to the fluorescent signal obtained from the housekeeping genes ribosomal phosphoprotein PO (RPLPo2), β-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for each sample.

Figure 1 . 2 -

 12 Figure 1. 2-DE of total cell extracts from HeLa (A) and HZR (B) cells. Two hundred

Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3. Low molecular weight part of 2-D electrophoretic gels of total HeLa (left) and

Figure 4 . 32 Figure 5 .

 4325 Figure 4. Effects of ER stress on the viability of HeLa and HZR cells in the presence of

Figure 6 .

 6 Figure 6. Real-time PCR of HPPD. Real-time quantitative, reverse transcriptase polymerase

Figure 7 .

 7 Figure 7. HPPD inhibition and cadmium toxicity in HeLa and HZR cells. (A) Parental

Figure 8 .

 8 Figure 8. Tyrosine and cadmium toxicity in HeLa and HZR cells. (A) Parental HeLa cells

Acknowledgements

This work was supported by a grant from the "Toxicologie Nucléaire et Environnementale" program. Dr David King (Swedish Orphan International AB, Stockholm, Sweden) is gratefully thanked for providing us with NTBC, as is Dr Pierre Richaud (CEA, IBEB, LB3M, Cadarache, France) for ICP-OES measurements.