Dr Fabienne Aujard
email: fabienne.aujard@wanadoo.fr

Florence Cayetanot

Jérémy Terrien

Eus J W Van Someren

Attenuated effect of increased daylength on activity rhythm in the old mouse lemur, a non-human primate

Keywords: Aging, Primate, Circadian rhythm, Locomotor activity, Rhythm stability, Body temperature, Daylength, Season

Adaptation of physiological and behavioral functions to seasonal changes in daylength is of major relevance for optimal fitness and survival. Because aging is characterized by changes in biological rhythms, it may be hypothesized that old animals fall short of showing a full adaptation to prolonged changes in the duration of daily light exposure, as naturally occurring in relation to season in younger individuals. To test this hypothesis, we analyzed changes in the patterns of daily locomotor activity and body temperature rhythms of young and old mouse lemurs (Microcebus murinus, Primates) exposed to short and long daylengths. The effect of an increase in the duration of daily light exposure was attenuated in old animals, as compared to younger lemurs. Although some age-related differences in the locomotor activity rhythm could be seen under exposure to short daylength, they were predominant under long daylength. Some mechanisms allowing adaptation to changing day length thus seem to be impaired at old age. Changes in coupling of circadian oscillators to the light-dark cycle and disturbances in the physiological responses to change in light duration should be further investigated.

INTRODUCTION

Aging is associated with changes in circadian rhythmicity of endocrine, metabolic and behavioral parameters in humans [START_REF] Weinert | Age-dependent changes of the circadian system[END_REF][START_REF] Van Someren | Live to the rhythm, slave to the rhythm[END_REF]. It has been demonstrated for various circadian rhythms that aging is characterized by a decrease in amplitude, a phase advance and an impaired resynchronization following abrupt phase shifts in the light-dark cycle. In humans, aging modifies the daily rhythm of restactivity. Aging is typically associated with (1) an increased fragmentation into shorter alternating periods of rest and activity; (2) an increased activity level during the major sleep period, (3) a decreased activity level during the major active period, and as a consequence (4) a decreased amplitude [START_REF] Witting | Effects of Aging on Entrainment and Rate of Resynchronization of Circadian Locomotor-Activity[END_REF][START_REF] Myers | Changes in circadian rhythms and sleep quality with aging: mechanisms and interventions[END_REF][START_REF] Huang | 76, 597-603. and locomotor activity as marker rhythms of aging of the circadian system in rodents[END_REF]. The amplitude, pattern and phasing of the 24-hour profile of body temperature also changes with aging [START_REF] Monk | Circadian Temperature Rhythms of Older-People[END_REF][START_REF] Duffy | Later endogenous circadian temperature nadir relative to an earlier wake time in older people[END_REF][START_REF] Gubin | The circadian body temperature rhythm in the elderly: Effect of single daily melatonin dosing[END_REF]. However, some of the agerelated changes can be ameliorated by increasing the exposure to artificial bright light [START_REF] Van Someren | Functional plasticity of the circadian timing system in old age: light exposure[END_REF]. Moreover, there is now some evidence that human rhythms are influenced by season through changes in the duration of daylength [START_REF] Bronson | Are humans seasonally photoperiodic?[END_REF], and it appears of interest to experimentally test the effect of daylength changes on the circadian rhythms of young and old subjects. However, changes in the duration of light exposure have rarely been taken into account in studies on circadian rhythms of old humans.

The effects of age on the circadian clock system have also been extensively studied in rodents (e.g. (Zee et al., 1992;[START_REF] Penev | Quantitative analysis of the age-related fragmentation of hamster 24-h activity rhythms[END_REF][START_REF] Valentinuzzi | Effects of aging on the circadian rhythm of wheel-running activity in C57BL/6 mice[END_REF]Mailloux et al., 1999;[START_REF] Zhang | Effects of photoperiod reduction on rat circadian rhythms of BP, heart rate, and locomotor activity[END_REF]), confirming rhythm disturbances such as an increased fragmentation and decreased amplitude of the daily locomotor activity rhythm, as well as a decreased precision in the onset of activity phase. Previous work in rodents and primates indicates that the sensitivity of the circadian timing system to short-term manipulation of light

A C C E P T E D

M A N U S C R I P T 4 exposure may decrease with age [START_REF] Zhang | Effects of aging on light-induced phase-shifting of circadian behavioral rhythms, fos expression and CREB phosphorylation in the hamster suprachiasmatic nucleus[END_REF][START_REF] Benloucif | Light-induced phase shifts of circadian activity rhythms and immediate early gene expression in the suprachiasmatic nucleus are attenuated in old C3H/HeN mice[END_REF][START_REF] Aujard | Artificially accelerated aging by shortened photoperiod alters early gene expression (Fos) in the suprachiasmatic nucleus and sulfatoxymelatonin excretion in a small primate, Microcebus murinus[END_REF]. Taken together, these data suggest that age could have profound effects on the synchronization of circadian rhythms to changes in daylength. Rodent studies have shown that the effect of photoperiod on the temporal organization of daily rhythms is modulated by age [START_REF] Scarbrough | Aging and photoperiod affect entrainment and quantitative aspects of locomotor behavior in Syrian hamsters[END_REF][START_REF] Benstaali | Effect of a short photoperiod on circadian rhythms of body temperature and motor activity in old rats[END_REF]. In primates, the importance of daylength in the expression of activity patterns [START_REF] Hill | Day length variation and seasonal analysis of behaviour[END_REF] and reproductive function [START_REF] Chik | Photoperiod-Driven Changes in Reproductive Function in Male Rhesus-Monkeys[END_REF] has been demonstrated in young adults only. It remains to investigate whether circadian rhythms responses to long-term changes in the duration of light exposure are comparable between old and younger primates.

The mouse lemur (Microcebus murinus, Primates) is a small nocturnal primate (body weight: 60 -90 g) originating from Madagascar. This primate exhibits photoperiod-dependent seasonal and daily rhythms in most of its biological functions. Daily exposure to light for longer than 12 h promotes sustained behavioral activity, whereas daily exposure to light for less than 12 h reduces behavioral activity and leads to an increase in fat deposits [START_REF] Perret | Environmental and social determinants of sexual function in the male lesser mouse lemur (Microcebus murinus)[END_REF]. The life span of this species is about 8 -10 years in captivity [START_REF] Perret | Change in photoperiodic cycle affects life span in a prosimian primate (Microcebus murinus)[END_REF]. Old animals show a decrease in amplitude of the seasonal variations in body mass, sexual hormones [START_REF] Aujard | Age-related effects on reproductive function and sexual competition in the male prosimian primate, Microcebus murinus[END_REF], melatonin [START_REF] Aujard | Artificially accelerated aging by shortened photoperiod alters early gene expression (Fos) in the suprachiasmatic nucleus and sulfatoxymelatonin excretion in a small primate, Microcebus murinus[END_REF] and DHEA-S (Perret andAujard, 2005, 2006). Moreover, a recent study demonstrated that thermoregulation differed according to photoperiod and age in this species [START_REF] Aujard | Behavioral thermoregulation in a non human primate: Effects of age and photoperiod on temperature selection[END_REF], see comments in [START_REF] Van Someren | Thermoregulation and aging[END_REF]. Several of these studies indicate that daily and seasonal biological rhythms are altered in old mouse lemurs in ways that are remarkably similar to what is observed in human aging [START_REF] Huang | 76, 597-603. and locomotor activity as marker rhythms of aging of the circadian system in rodents[END_REF][START_REF] Cayetanot | Shortened seasonal photoperiodic cycles accelerate aging of the diurnal and circadian locomotor activity rhythms in a primate[END_REF]: a decrease in amplitude of the locomotor activity level, an increased activity during the resting diurnal phase and an increase in fragmentation. For these and other reasons, the mouse lemur can be regarded as a model of primary importance for human aging [START_REF] Bons | Microcebus murinus: a useful primate model for human cerebral aging and Alzheimer's disease?[END_REF].

A C C E P T E D M A N U S C R I P T 5
In the present study, we examined whether a prolonged exposure to an increased duration of daily light exposure would equally affect younger and older mouse lemurs with respect to their locomotor activity and body temperature rhythms. If a differential response to changes in the duration of daily light exposure exists, age-related differences in the expression of daily rhythms observed under one specific photoperiod may not surface under another photoperiod.

METHODS

Animals

All animals used in this study were born and kept in captivity in the laboratory breeding colony of the CNRS at Brunoy (MNHN, France, license approval N° A91.114.1). General conditions of captivity were maintained constant with respect to ambient temperature (24 -26 °C) and relative humidity (55%), food and water available ad libitum (including fresh fruits, a milky mixture and meal worms). In captivity, seasonal variations of physiological functions can be entrained by alternating 6-month periods of long daylength (14 h of light/day) and short daylength (10 h of light/day) under artificial light. Nine young male mouse lemurs (mean age: 2.5 ± 0.5 years, range: 1 -4 years) and 8 old male mouse lemurs (mean age: 7.3 ± 0.8 years, range: 6 -10 years) were studied.

Body mass of animals was higher in short daylength than in long daylength (t-test, t (16) = 2.92, P < 0.01). The body mass did not differ significantly between the 2 groups of mouse lemurs under each photoperiod (short days: young: 109 ± 4 g, old: 106 ± 5 g, t-test, t(15) = 0.5, NS; long days: young: 93 ± 9 g, old: 91 ± 4 g, t-test, t(15) = 0.17, NS). Thus, the age related differences observed in this study could not be attributed to differences in body mass. Cages were placed in a chronobiological animal facility (Type A130-SP, Fisher Scientific Bioblock, Illkirch, France) with filtered air and controlled ambient temperature (25 ± 1 °C).

Each separated compartment was equipped with independent light-dark cycles, provided by cool fluorescent lamps (250 -380 lux) during the light phase and with a dim red light (0.002 lux) during the dark phase. The lamps were placed on the side of the cage, at 10 cm from a wooden nest, where the animal could retire during the entire sleeping period, until it started its activity. All animals were tested after at least 1 month of exposure to a given photoperiod, a time lapse necessary for their complete physiological adaptation to the photoperiod [START_REF] Perret | Environmental and social determinants of sexual function in the male lesser mouse lemur (Microcebus murinus)[END_REF]. The recording period began at least 3 weeks after surgery. Locomotor activity and T c were recorded simultaneously during 14 days after at least one month of exposure to a lightdark (LD) cycle of 10 h of light per day (short daylength), and then after at least one month of exposure to a LD cycle of 14 h of light per day (long daylength). The change in photoperiod was done by advancing the light onset by 4 h, as previously used in synchronization studies [START_REF] Perret | Daily hypothermia and torpor in a tropical primate: synchronization by 24-h light-dark cycle[END_REF].

Data analysis

Data were collected using a computerized data acquisition system (LabPro v.3.11, Data Sciences, St Paul, Minnesota, USA). T c (in °C) was recorded for 10 s every 5 min and locomotor activity (in arbitrary units, AU) was recorded continuously. Actograms were performed using the Clocklab software (Actimetrics Inc., Evanston, IL). Quantitative characteristics of the locomotor activity rhythm were determined with non-parametric methods as previously described [START_REF] Van Someren | Bright light therapy: Improved sensitivity to its effects on rest-activity rhythms in Alzheimer patients by application of nonparametric methods[END_REF][START_REF] Huang | 76, 597-603. and locomotor activity as marker rhythms of aging of the circadian system in rodents[END_REF]. Briefly, the following variables were calculated from the locomotor activity data. The interdaily stability quantifies the regularity of the timing of onset and offset of activity bouts and of the specific individual activity profile over days (increasing stability from 0 to 1). The intradaily variability quantifies the fragmentation of the period of rest and activity (increasing fragmentation from 0 to 2). L5 quantifies activity during the major sleep period as the average activity during the most restful 5-hour period of the activity profile. M10 similarly quantifies the average activity during the 10 most active hours. The amplitude of the locomotor activity rhythm was calculated as the difference between L5 and M10 values divided by their sum.

Activity onset was defined as the first 6 successive bins where activity was greater than the mean locomotor activity during the light phase. T c data were analyzed for average nocturnal T c and both the level and timing of the daytime T c minimum (expressed in minutes prior to the time of lights on). When the activity onset precedes lights off, this phase advance is expressed by a positive value. Inversely, a phase delay is expressed by a negative value.

All values are expressed as mean ± SEM and analyses were conducted using SYSTAT for Windows. Data were analyzed by two-way analysis of variance (ANOVA) on the effects of age, duration of light exposure and their interaction on repeated measures. One-way ANOVA was used to test the effect of duration of light exposure on activity onset in each age category. Differences in which p < 0.05 were considered to be significant. 1. We found a significant effect of the duration of light exposure, no overall effect of age but a significant age x daylength effect on interdaily stability, indicating that an increase in the duration of light exposure enhanced interdaily stability less in old than in young lemurs (Fig. 2A). There was a significant effect of the duration of light exposure and a significant effect of age on intradaily variability (Fig. 2B).

Young animals exposed to long daylength exhibited a lower level of intradaily variability of the locomotor activity rhythm compared to young mouse lemurs exposed to short photoperiod. The significant age x daylength effect indicated that the increase in the duration of light exposure attenuated intradaily variability in young, but not in old animals.

The activity rhythm amplitude was also significantly affected by the duration of light exposure (Fig. 2C). There was a main effect of age and a significant age x daylength effect: an increase in the duration of light exposure enhanced the amplitude of the locomotor activity

A C C E P T E D M A N U S C R I P T

9 rhythm in young, but not in old animals. The activity level during the major sleep period (L5) was significantly affected by the duration of light exposure (Fig. 2D) as well. Due to high interindividual variability, the effect of age was only close to significance and no significant effect of age x daylength could be found. However, an increase in the duration of light exposure suppressed L5 in young mouse lemurs, but had no significant effect in old animals, which exhibited a high level of L5 whatever the duration of daily light exposure. The amount of activity during the major wakefulness period (M10) was significantly affected by the duration of light exposure, age and their interaction (Fig. 2E): when exposed to long daylength, young animals exhibited an increase of 43 ± 7 % relative to their M10 level when exposed to short daylength, while the average increase in the age group was only 26 ± 7 %.

The onset phase of the major activity period always preceded lights off. There was no overall effect of the duration of daily light exposure on the onset of activity, but there was a significant effect of age (Fig. 2F). In all cases, animals started their activity period just shortly before lights off, except for old animals exposed to long daylength which exhibited a considerably earlier onset of their activity compared to their onset of activity under short daylength exposure. This effect of the duration of daily light exposure was significant in old animals, but not in younger ones (Table 2). Representative actograms of a young and an aged mouse lemurs exposed to long daylength are illustrated in Fig. 3.

Effect of the duration of daily light exposure on the core body temperature rhythm of young and old mouse lemurs

There was no age effect on the nocturnal level of T c or on the minimum T c value (Fig.

4A-B)

. A significant effect of the duration of daily light exposure was found for these two parameters that was due to an increase in average nocturnal T c and in the minimum of T c from short daylength to long daylength in both young and in old animals.

A C C E P T E D M A N U S C R I P T 10

There was a significant effect of age on time of occurrence of the minimum of T c (Fig. 4C). Whatever the duration of daily light exposure, the time of occurrence of the minimum of T c was delayed in old animals compared to younger ones. The minimum of T c occurred on average 174 ± 25 min after lights on in young and 280 ± 23 min after lights on in old animals, representing a delay of almost 2 h for old animals compared to younger ones.

DISCUSSION

An increase in the duration of daily light exposure had a more pronounced effect on the 24-h profile of locomotor activity rhythm in young than in old mouse lemurs. In old , as compared to short days, exposure to long days led to an increase in interdaily stability and activity level during the major period of wakefulness, yet in both cases less than the respective increases found in younger animals. Whether age affected the locomotor activity rhythm thus depended to a large extent on whether animals were observed during short days or long days: age-differences were much more pronounced during the period of longer daily light exposure. By contrast, no age difference could be detected in the response of the body temperature rhythm to changes in the duration of daily light exposure. Thus, although the activity rhythm response to an increase in the duration of daily light exposure was less in old animals, it was not completely absent, and even intact for the temperature rhythm response.

In young mouse lemurs, an increase in the duration of daily light exposure induced an increase in amplitude and activity during the major active period, and a decrease in activity during the major restful period, compared to exposure to short daylength. These effects do not match with the prediction of a potential masking effect of an increase in the duration of light exposure on the locomotor activity pattern of a nocturnal species, and rather suggest a full

A C C E P T E D M A N U S C R I P T

11 adaptation to each daylength. In aged animals, the effects of an increase in the duration of daily light exposure on locomotor activity levels were attenuated.

Like in young mouse lemurs, old animals exhibited a higher interdaily stability of their locomotor activity rhythm when exposed to longer days. A similar finding was observed in the elderly, in demented patients only [START_REF] Van Someren | Circadian rest-activity rhythm disturbances in Alzheimer's disease[END_REF]. The patients showed a relatively higher interdaily stability during the summer. This indicates that there is at least some preservation of functional plasticity of the circadian timing system at high age (for review see [START_REF] Van Someren | Functional plasticity of the circadian timing system in old age: light exposure[END_REF]). Moreover, it is noteworthy that old animals started at higher interdaily stability during the shorter days than young animals did. A higher interdaily stability has also been observed in elderly humans and has been proposed to reflect a more rigid schedule of activity as an adaptive response to overcome a circadian system that is more easily disrupted [START_REF] Monk | Daily Social Rhythms in the Elderly and Their Relation to Objectively Recorded Sleep[END_REF]Carvalho-Bos et al., 2007;[START_REF] Van Someren | Live to the rhythm, slave to the rhythm[END_REF]. ().

Whatever the duration of the daily light exposure, young mouse lemurs were tightly entrained to the onset of lights off. In old animals, a long photoperiod was associated with an advanced onset of activity. Under short days, no difference could be seen between young and old mouse lemurs, since all animals started their activity close to the time of lights off. In young mouse lemurs maintained in free running condition (constant dim red light), the subjective day lasted 9.5 ± 0.5 hr [START_REF] Perret | Daily hypothermia and torpor in a tropical primate: synchronization by 24-h light-dark cycle[END_REF]. It could thus be argued that the endogenous architecture of the circadian system would more readily adapt to a light period of 10 than of 14 hr. According to this contention, a 10-hr light period would require only an additional extension of 0.5 hr on top of the endogenous 9.5 hr of subjective day, while a 14-hr light period would require an additional 4.5 hr extension for an efficient synchronization. A reduced strength of lights on and lights off as potential zeitgebers has been demonstrated in old mice [START_REF] Weinert | The relative Zeitgeber strength of lights-on and lights-off is changed in old mice[END_REF]. Although this remains to be demonstrated in the In contrast to the activity rhythm response, the body temperature rhythm response to an increased duration of daily light exposure was preserved in old mouse lemur. The timing of minimal body temperature was delayed in old mouse lemurs compared to younger ones, irrespective of photoperiod. Of note, the delayed body temperature minimum in old mouse lemurs contrasts with the phase advance typical of old humans [START_REF] Monk | Circadian Temperature Rhythms of Older-People[END_REF][START_REF] Duffy | Later endogenous circadian temperature nadir relative to an earlier wake time in older people[END_REF]. On the other hand, a delay in body temperature has been observed in demented elderly subjects [START_REF] Harper | Differential circadian rhythm disturbances in men with Alzheimer disease and frontotemporal degeneration[END_REF], and Duffy et al. [START_REF] Duffy | Later endogenous circadian temperature nadir relative to an earlier wake time in older people[END_REF] demonstrated that the body temperature nadir was actually delayed when expressed relative to the waking onset in the elderly. In mouse lemurs, the body temperature minimum was affected by photoperiod but not by age, while the timing of minimal body temperature changed according to age but not photoperiod. This corroborates the results of Benstaali et al. [START_REF] Benstaali | Effect of a short photoperiod on circadian rhythms of body temperature and motor activity in old rats[END_REF] in rats, showing that age and photoperiod acted independently on the daily rhythm of body temperature. The preservation of the body temperature rhythm response to daylength in old animals stands out as compared to the attenuated activity rhythm response, and also compared to previous findings in other rhythms. A decreased amplitude in seasonal variations of several other physiological parameters has also been demonstrated in old mouse lemurs, such as body weight [START_REF] Aujard | Artificially accelerated aging by shortened photoperiod alters early gene expression (Fos) in the suprachiasmatic nucleus and sulfatoxymelatonin excretion in a small primate, Microcebus murinus[END_REF], behavioral thermoregulation [START_REF] Aujard | Behavioral thermoregulation in a non human primate: Effects of age and photoperiod on temperature selection[END_REF], testosterone [START_REF] Aujard | Age-related effects on reproductive function and sexual competition in the male prosimian primate, Microcebus murinus[END_REF] and DHEA-S [START_REF] Perret | Aging and season affect plasma dehydroepiandrosterone sulfate (DHEA-S) levels in a primate[END_REF].

In summary, although some age-related differences in the locomotor activity rhythm could be seen under exposure to short daylength, they were predominant under long daylength. Under short days, the patterns of old animals were close to those observed in young animals. Daylength effects on the rhythm parameters of young -but not old -animals led to the emergence of various age-related differences under exposure to long days. Some

  carried out in accordance with the European Communities council Directive (86/609/EEC). All efforts were made to minimize animal suffering throughout the experiments.Procedures and lighting conditionsLocomotor activity and core body temperature (T c ) rhythms were recorded by telemetry (Dataquest LabPro System, Data Sciences, St Paul, Minnesota, USA) after implantation of a radio sensor-transmitter (TA10TA-F20 Data Sciences, St Paul, Minnesota, USA) in the visceral cavity under anesthesia (Valium 2mL/100g intra muscular, Imalgen 2mL/100g intra peritoneal). Mouse lemurs were housed individually in a cage containing a receiver board.

  duration of daily light exposure on the locomotor activity rhythm of young and old mouse lemurs Average 48h-profiles of locomotor activity and T c of young and old mouse lemurs exposed to either short or long daylength are represented in Fig. 1. Whatever their age category, all animals exhibited a daily rhythm of both parameters. In all cases, the nocturnal period corresponded to the active phase of the day, where animals exhibited the highest level of locomotor activity and the highest T c values. The profiles differed according to the duration of light exposure and age. Daylength-induced effects on the quantitative parameters of the daily locomotor activity rhythm in young and old mouse lemurs are represented in Fig. 2 and statistical results are indicated in Table

  such an age-related alteration may also explain the photoperiod-dependent change in activity onset in old mouse lemurs.

  synchronization and entrainment to changing day length seem to be impaired with aging. It may be that at high age, a higher intensity of environmental light is required in order to obtain a full behavioral circadian response. It is also possible that, by increasing daylength, the coupling of individual circadian oscillators to the LD cycle or to other circadian parameters is modified. Changes in coupling of circadian oscillators to the light-dark cycle and disturbances in the physiological responses to change in light duration should be further investigated.

Figure captions Fig. 1 .

 captions1 Figure captions

Fig. 2 .

 2 Fig. 2. Differences in quantitative parameters of the locomotor activity rhythm (A: interdaily

Fig. 3 .

 3 Fig. 3. Representative actograms of the daily locomotor activity rhythm of a young and an old

Fig. 4 .

 4 Fig. 4. Differences in the daily body temperature (T c ) rhythm parameters (A: nocturnal T c , B:

F

Table 1 .

 1 Two-way ANOVA results on the effect of age and daylength on the daily rhythms of locomotor activity and body temperature (T c ) in young and old mouse lemurs exposed to either short (LD 10:14) or long photoperiod (LD 14:10). L5: activity during the major resting period of the day; M10: activity during the major active period of the day.

	p

p F p F

Table 2 .

 2 One-way ANOVA results on the effect of daylength on the onset of locomotor activity in each age category (young or old mouse lemurs), when exposed to either short(LD 10:14) or long photoperiod (LD 14:10).

FIG.3

ACKNOWLEDGEMENTS

This work was supported by grants from the European Community QLK6-CT-2002-02258, ACI Neurosciences, Institute for Longevity, Foundation for Medical Research and (EvS) VIDI Innovation Grant 016.025.041, Netherlands Organization for Scientific Research (NWO), The Hague, The Netherlands.

DISCLOSURE STATEMENT

There is no conflict of interest concerning this work. Full agreement was obtain to conduct this work on the breeding colony of Brunoy (agreement # A91.114.1).

A C C E P T E D

M A N U S C R I P T


Manuela Casadei

Tiziana Persichini

Fabio Polticelli

Giovanni Musci

Marco Colasanti
email: colasant@uniroma3.it

S-glutathionylation of metallothioneins by nitrosative/oxidative stress

Keywords: diamide, metallothioneins, nitric oxide, nitrosative/oxidative stress

Cystein residues within metallothionein (MT) structure have been shown to be particularly prone to S-nitrosylation. The objective of this study was to examine the possibility that MTs undergo S-glutathionylation under nitrosative/oxidative stress. MT from rabbit liver was treated with different concentrations of GSNO, diamide plus GSH or H 2 O 2 plus GSH. Parallel sets of samples were treated with 10 mM DTT for 30 min at 37°C to reduce mixed disulphides. Incubations were then processed for Western blot or dotimmunobinding assay. Western blot with anti-MT or anti-GSH were also performed on peripheral blood mononuclear cell extracts. Structural aspects of S-glutathionylation of MTs were also examined. Treatment with GSNO, diamide/GSH or H 2 O 2 /GSH induced a dosedependent increase in the levels of MT S-glutathionylation. This effect was completely reversed by treatment with the reducing agent DTT, indicating that S-glutathionylation of MT protein was related to formation of protein-mixed disulphides. Structural analysis of rat MT indicated that Cys residues located in the N-terminal domain of the protein are the likely targets for S-glutathionylation, both for their solvent accessibility and electrostatics induced reactivity. S-glutathionylation of MT, given its reversibility, would provide protection from irreversible oxidation of Cys residues, thus representing a mechanism of high potential biological relevance.

Introduction

Nitric oxide (NO) is a signaling molecule involved in several diverse physiological and pathological processes. Synthesized from L-arginine by three isoforms of the enzyme nitric oxide synthase (NOS) (Moncada and Palmer, 1991), NO is now known to control vascular smooth muscle tone, to inhibit platelet and inflammatory cell adhesion and activation, and to be a transmitter at non-adrenergic non-cholinergic synapses (Moncada et al., 1991;[START_REF] Quinn | Nitric oxide: an endogenous gas[END_REF]. NO has been also revealed to modulate apoptosis, in a variety of cell types, including human inflammatory cells [START_REF] Taylor | Nitric oxide: a key regulator of myeloid inflammatory cell apoptosis[END_REF].

The wide variety of effects of NO is achieved, among others, through its specific interactions with cysteine-containing proteins [START_REF] Stamler | Nitrosylation. the prototypic redox-based signaling mechanism[END_REF]. The reversible attachment of NO to thiols, a covalent modification known as S-nitrosylation, represents most of the effects of NO on the activity of proteins (with the exclusion of metal-containing proteins). It has often been suggested that S-nitrosothiols represent a more stable metabolite of NO that can either be stored or transported, although the evidence for this is sparse [START_REF] Hogg | The biochemistry and physiology of S-nitrosothiols[END_REF]. The abundance of GSH (0.5 to 10 mM) in the intracellular compartment renders the formation of S-nitrosoglutathione (GSNO), structurally the simplest S-nitrosothiol, kinetically favorable [START_REF] Singh | Mechanism of nitric oxide release from S-nitrosothiols[END_REF]. GSNO can promote S-glutathionylation, i.e., the formation of mixed disulfides between protein thiol and GSH [START_REF] Giustarini | S-Nitrosation versus S-Glutathionylation of Protein Sulfhydryl Groups by S-Nitrosoglutathione[END_REF]. Otherwise, Sglutathionylated proteins may be generated following the protein thiol groups activation by oxidative/nitrosative modifications to give thiyl radical, sulphenic acid, or protein Snitrosothiol/S-nitrosylated protein [START_REF] Giustarini | Sglutathionylation: from redox regulation of protein functions to human diseases[END_REF]. These modifications may be stable as such or react with GSH to form mixed disulphides (i.e., S-glutathionylated proteins). All these modifications are reversible and can be reverted by an increase in the GSH/GSSG ratio, reduced thiols, or enzymatic reactions [START_REF] Giustarini | Sglutathionylation: from redox regulation of protein functions to human diseases[END_REF]. Evidence suggests that protein S-glutathionylation, being characterized by sensitivity, specificity and reversibility, might be a common mechanism for the global regulation of protein functions in different cellular types [START_REF] Giustarini | Sglutathionylation: from redox regulation of protein functions to human diseases[END_REF][START_REF] Klatt | Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress[END_REF]. In particular, it has been focused the role of S-glutathionylation as a mechanism of redox-mediated signal transduction as well as an adaptive cellular response protecting critical regulatory proteins from permanent loss of function, as a consequence of oxidative and nitrosative stress [START_REF] Klatt | Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress[END_REF].

Metallothioneins (MTs) are a family of small proteins (6-7 kDa) with a high content of Cys residues (20 cysteines located in highly conserved positions) [START_REF] Kagi | Biochemistry of metallothionein[END_REF][START_REF] Vasak | Metallothioneins: new functional and structural insights[END_REF], which allows these proteins to coordinate multiple mono (Cu + , Ag + ) or divalent metals (Zn 2+ , Cd 2+ ). Interestingly, NO has been shown to induce S-nitrosylation of Cys residues of intracellular MTs and consequent Zn 2+ or Cd 2+ release both in vitro [START_REF] Aravindakumar | Nitric oxide induces Zn2+ release from metallothionein by destroying zinc-sulphur clusters without concomitant formation of S-nitrosothiol[END_REF][START_REF] Kroncke | Nitric oxide destroys zinc-sulfur clusters inducing zinc release from metallothionein and inhibition of the zinc finger-type yeast transcription activator LAC9[END_REF][START_REF] Misra | Evidence that nitric oxide enhances cadmium toxicity by displacing the metal from metallothionein[END_REF][START_REF] Zangger | Nitric oxide selectively releases metals from the amino-terminal domain of metallothioneins: potential role at inflammatory sites[END_REF] and in vivo [START_REF] Katakai | Nitric oxide induces metallothionein (MT) gene expression apparently by displacing zinc bound to MT[END_REF]Pearce et al., 2000b;Pearce et al., 2000a;[START_REF] Schwarz | Metallothionein protects against the cytotoxic and DNA-damaging effects of nitric oxide[END_REF]. It is tempting to speculate that the same "activated" Cys residues of MTs are highly reactive to S-glutathionylation as well.

In this study, we examined the possibility that MTs undergo S-glutathionylation under nitrosative/oxidative stress. To this purpose, we exposed the MT to GSNO and other oxidizing agents (diamide or hydrogen peroxide in the presence of GSH), and performed Western blot and dot-immunobinding assay. Structural aspects of S-glutathionylation of MTs were also examined.

Materials and methods

Chemicals and reagents

Metallothionein (MT) from rabbit liver, bovine serum albumin (BSA), Nethylmaleimide (NEM), and protease inhibitors (leupeptin, phenylmethylsulfonyl fluoride and protease inhibitor cocktail) were purchased from Sigma-Aldrich (Milan, Italy). A&G-Sepharose beads and protein molecular marker were from Amersham Biosciences (Uppsala, Sweden). The redox reagents reduced L-glutathione (GSH), diamide, hydrogen peroxide (H 2 O 2 ) and 2,4-dithiothreitol (DTT) were also obtained from Sigma-Aldrich (Milan, Italy).

Fresh solutions of GSH and diamide were bubbled with N 2 and stored in aliquots at -80°C.

To avoid spontaneous oxidation of GSH and diamide, aliquots were thawed only once and the unused portion was discarded. Mouse anti-GSH monoclonal antibody was from Virogen (Watertown, MA, USA). Mouse anti-MT monoclonal antibody was purchased from Stressgen (Assay Designs, Inc. Michigan, Ann Arbor, MI, USA) and rabbit anti-MT polyclonal antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Horseradish peroxidase-conjugated anti-mouse IgG was from Calbiochem (La Jolla, CA, USA). All other reagents were of analytical grade from commercial sources.

Synthesis of GSNO

S-nitrosoglutathione (GSNO) was synthesized by combining equimolar concentrations (200 mM) of GSH and NaNO 2 in 0.5 M HCl at room temperature. After 5 min of incubation, the solution was neutralized by addition of NaOH (200 mM). Stock solutions of GSNO were bubbled with N 2 and stored in aliquots at -80°C.

Diamide, H 2 O 2 and GSNO treatment of MT

MT from rabbit liver (4 µg/assay) was diluted in lysis buffer (50 mM Tris-HCl pH 7.4, 2 mM EGTA, 2 mM EDTA, 1% (v/v) Triton X-100 and protease inhibitors). MT was then treated with different concentrations of GSNO, diamide plus GSH or H 2 O 2 plus GSH, for 30 min at 37°C. Reactions were stopped by addition of 5 mM NEM to block unreacted thiol groups, and mixtures were incubated for additional 30 min at 37°C. Parallel sets of samples were co-treated with 10 mM DTT for 30 min at 37°C, to reduce mixed disulphides.

Incubations were then processed for Western blot or dot-immunobinding assay as described below.

Immunoprecipitation of MT

Peripheral Blood Mononuclear Cells (PBMC) from voluntary healthy subjects were rinsed in cold PBS and lysed in lysis buffer supplemented with 50 mM NEM. Cell lysates (50 µg) were incubated with 10 µl of polyclonal antibody raised against MT (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4°C overnight on a rocker platform.

Immunoprecipitates were adsorbed to protein A&G-Sepharose at 4°C for 2 h, collected by centrifugation at 270 × g for 10 min at 4°C, washed three times with lysis buffer, and eluted from beads with 5% SDS. Samples were then analyzed by non-reducing SDS-PAGE as described below.

Identification of S-glutathionylated MT by Western Blot

Protein samples were dissolved in a non-reducing sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 5% SDS, 0.05% bromophenol blue) supplemented with 5 mM NEM, were denatured at 100°C for 5 min, and were separated on a 15% sodium dodecyl sulphatepolyacrylamide gel. In a parallel set of samples, 10 mM DTT was added to the sample buffer.

After electrophoresis, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and nonspecific binding was blocked by incubation in 3% BSA diluted in TBST.

Membranes were then probed with primary monoclonal antibodies against MT or GSH, diluted 1:1000 in blocking buffer. After washing, blots were incubated with secondary antibodies conjugated to horseradish peroxidase (1:15000 dilution in blocking solution).

Protein-antibody reactions were detected with an ECL kit (Amersham Biosciences). For the quantification of S-glutathionylation levels, films were digitally scanned and scans were analyzed with the Multi-Analyst PC version 1.1 program (Bio-Rad Laboratories S.r.l., Milan, Italy).

Identification of S-glutathionylated MT by Dot-immunobinding assay

Protein samples were diluted in a denaturing solution (62.5 mM Tris-HCl pH 7.5, 0.05% (w/v) SDS) without reducing agents, containing 5 mM NEM. In a parallel set of samples 10 mM DTT was added to the denaturing solution. Samples were then subjected to dot-immunobinding assay. Immunodetection of protein S-glutathionylation was performed as described for Western blot analysis. Estimates of the relative S-glutathionylation levels were obtained dividing the optical density of the glutathionylated MT spot by the optical density of the MT spot (Bio-Rad Multianalyst). The statistical analysis of the data for single comparisons was performed by Student's t test.

Structural analysis

Structural determinants of Cys residues reactivity have been analyzed using rat liver MT three-dimensional structure (PDB code 4MT2) [START_REF] Braun | Comparison of the NMR solution structure and the x-ray crystal structure of rat metallothionein-2[END_REF]. The accessible surface area of Cys Sγ atoms has been calculated using Michael Sanner's MSMS program (available at http://www.scripps.edu/~sanner/), with a 1.4 Å probe size.

Results

To verify whether MT is susceptible to S-glutathionylation under oxidative/nitrosative stress conditions, we exposed commercially available MT from rabbit liver to GSNO, and to other oxidants (i.e., diamide or H 2 O 2 , in the presence of GSH).

MT was first treated with varying concentrations of GSNO (0, 0.25, 0.5, 1, 2, and 5 mM) and tested by Western blot analysis using monoclonal antibodies raised against GSH or MT to probe the membrane. Results shown in Fig. 1A,C suggest that the appearance of Sglutathionylated MT induced by the oxidative stress condition correlates with the increasing GSNO concentration, the maximal effect being observed at 2 mM nitrosothiol.

Glutathionylated MT was found to run mainly as a 14 kDa band (Fig. 1A), with a minor portion running at 28 kDa at the highest GSNO concentration (data not shown). When the membrane was probed with an anti-MT antibody, a 6-7-kDa immunoreactive band was mainly detected in untreated MT (Fig. 1B, lane 1) as well as in GSNO-treated MT (Fig. 1B, lanes 2-6), consistent with the reported molecular weight for the monomeric form of MT.

Interestingly, the treatment with GSNO induced the progressive appearance of lower mobility immunoreactive bands, with molecular masses (multiple of 6-7 kDa) apparently corresponding to different oligomeric forms of MT . As shown by the densitometric analysis in Fig. 1D, the MT dimer running at 14 kDa was the main product. These results are consistent with the well-known tendency of MT protein to form stable complexes and aggregates under oxidative stress conditions [START_REF] Hou | Cysteine-independent polymerization of metallothioneins in solutions and in crystals[END_REF][START_REF] Oppermann | Identification and spatial analysis of metallothioneins expressed by the adult human lens[END_REF][START_REF] Suzuki | Isolation and identification of metallothionein isoforms (MT-1 and MT-2) in the rat testis[END_REF][START_REF] Vallee | Zinc fingers, zinc clusters, and zinc twists in DNA-binding protein domains[END_REF][START_REF] Whitacre | Application of Western blotting to the identification of metallothionein binding proteins[END_REF][START_REF] Wilhelmsen | Evidence for oligomerization of metallothioneins in their functional state[END_REF], and suggest that S-glutathionylation mainly provokes dimerization of MT. In order to validate the results obtained by SDS-PAGE (Fig. 1A-D) and actually to show at once the total amount of immunoreactive MT, a simpler analysis of MT glutathionylation could be performed by using a dot blot procedure. Again, MT was treated with varying concentrations of GSNO. After 30 min of incubation samples were treated with either NEM, to block residual free thiols, or with DTT, to reduce any S-thiolated cystein. Detection of S-glutathionylated MT was obtained analyzing samples treated with GSNO by dot-immunobinding assay using monoclonal antibodies raised against GSH or MT. As expected, results (shown in Fig. 1E,G) were similar to those obtained by Western blot, in that the treatment with GSNO induced progressively higher levels of MT S-glutathionylation. To investigate whether GSNO was inducing the formation of protein-mixed disulphides on MT, samples were treated with the reducing agent DTT after exposure to GSNO. As shown in Fig. 1F, 10 mM DTT was able to completely reverse S-glutathionylation, as demonstrated by the disappearance of the immunostained spots, suggesting that mixed disulfides are formed in the presence of GSNO.

Next, MT was treated with different concentrations of the highly specific thiol oxidant diamide (0, 0.1, 0.2, 0.5, 1, and 2 mM) and of the physiologically occurring oxidant H 2 O 2 (0, 0.2, 0.5, 1, 2, and 5 mM), both in the presence of 1 mM GSH. Again reversibility was assessed by addition of the sulfhydryl-reducing agent DTT. Consistent with data obtained with GSNO treatment, both diamide (Fig. 2A,B) and H 2 O 2 (Fig. 2C,D) were able to induce a dose-dependent increase of S-glutathionylated MT in the presence of GSH. Again, 10 mM DTT completely reversed S-glutathionylation of MT induced by diamide and H 2 O 2 , as demonstrated by the disappearance of the immunostained spots (Fig. 2E and 2F, respectively).

Data presented so far demonstrate that S-glutathionylation of MT can be induced in vitro by a number of nitrosating/oxidizing mixtures. The question now is whether MT can be found glutathionylated in vivo. To address this point, PBMC lysates were immunoprecipitated with a polyclonal antibody raised against MT, and immunoprecipitates were analyzed by Western blot. As shown in Fig. 3A, MT was found to be highly aggregated under our experimental conditions. Interestingly, MT was also reactive toward the anti-GSH antibody (Fig. 3B), suggesting that in vivo it is heavily glutathionylated.

The molecular basis of susceptibility of rat MT to S-glutathionylation can be rationalized through comparison with the other well known modification of Cys residues, i.e., Snitrosylation. It is well known that the structural environment of the NO-reactive cysteine Sγ atom is critical for the S-nitrosylation [START_REF] Ascenzi | Re-evaluation of amino acid sequence and structural consensus rules for cysteine-nitric oxide reactivity[END_REF]. In fact, the solvent accessibility of the thiol group largely determines cysteine reactivity. In addition, the presence of basic residues in the vicinity of Cys residues facilitates deprotonation of the thiol group, thus increasing its reactivity. Similar considerations can be applied to glutathionylation reactions which are dependent as well on the reactivity of the cysteine thiol group. With this concepts in mind, rat liver MT three-dimensional structure has been analyzed (PDB code 4MT2) [START_REF] Braun | Comparison of the NMR solution structure and the x-ray crystal structure of rat metallothionein-2[END_REF] to uncover structural characteristics that allow to hypothesize the likely glutathionylation sites. First, the accessible surface area of Cys residues Sγ atom has been measured. As can be seen from Table 1 and Fig. 4, only for three Cys residues the measured values are significantly different from zero: Cys7, Cys13 and Cys59. Two out of these three residues (i.e., Cys7 and Cys13) are located in the N-terminal domain of MT which has been previously shown to be the selective target of S-nitrosothiol formation [START_REF] Zangger | Nitric oxide selectively releases metals from the amino-terminal domain of metallothioneins: potential role at inflammatory sites[END_REF].

Then, MT structure has been inspected to analyze the structural microenvironment of Cys residues as far as the presence of basic and acidic residues is concerned. From Fig. 4 it can be seen that two Cys residues located in the N-terminal domain of MT, Cys19 and Cys21, are less than 5 Å away from two Lys residues, Lys31 and Lys22. No other cysteine residue has charged residues in its immediate structural neighborhood. Based on the above considerations it seems likely that, as observed for nistrosylation of Cys residues of MT [START_REF] Zangger | Nitric oxide selectively releases metals from the amino-terminal domain of metallothioneins: potential role at inflammatory sites[END_REF], the target for S-glutathionylation is represented by MT N-terminal domain Cys residues.

Discussion

Oxidative/nitrosative stress conditions may cause chemical modifications on sensitive proteins. Such modifications, if irreversible, are usually associated with permanent loss of function and may lead to the elimination or to the accumulation of the damaged proteins. It has been proposed that reversible modifications, in particular at the Cys residues as Sglutathionylation, may have a dual role of protection from cysteine irreversible oxidation and modulation of protein function. The molecular mechanisms of protein function regulation by S-glutathionylation in response to oxidative and nitrosative stress have been previously reviewed by [START_REF] Klatt | Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress[END_REF]. They put special attention to the role of S-glutathionylation in stress signaling pathways and in the adaptive cellular response to oxidative and/or nitrosative insult. In particular, they focus on the role of S-glutathionylation as a mechanism of redox-mediated signal transduction as well as an adaptive cellular response protecting critical regulatory molecules from permanent loss of function, as a consequence of oxidative and nitrosative stress. Note that the accumulation of oxidatively irreversibly modified proteins (e.g., carbonylated proteins) is an important hallmark of biological ageing and pathological situations that are associated with oxidative/nitrosative stress [START_REF] Brigelius | Identification and quantitation of glutathione in hepatic protein mixed disulfides and its relationship to glutathione disulfide[END_REF][START_REF] Giustarini | Sglutathionylation: from redox regulation of protein functions to human diseases[END_REF][START_REF] Stadtman | Role of oxidant species in aging[END_REF][START_REF] Stadtman | Reactive oxygen-mediated protein oxidation in aging and disease[END_REF].

Although the biological function(s) of MTs still remain somewhat elusive [START_REF] Palmiter | The elusive function of metallothioneins[END_REF], they have been proposed to act as radical scavengers under oxidative stress [START_REF] Maret | Metallothionein/disulfide interactions, oxidative stress, and the mobilization of cellular zinc[END_REF][START_REF] Sato | Oxygen free radicals and metallothionein[END_REF][START_REF] Thornalley | Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals[END_REF]. It has been also reported that MTs play an important role in heavy metal detoxification [START_REF] Cherian | Role of metallothionein in carcinogenesis[END_REF][START_REF] Hamer | Metallothionein[END_REF]) and zinc homeostasis [START_REF] Kagi | Biochemistry of metallothionein[END_REF][START_REF] Li | Ligand substitution reactions of metallothioneins with EDTA and apo-carbonic anhydrase[END_REF]. The tight metal binding in the α -domain has been hypothesized to be important for the detoxification of heavy metals, whereas the function of MTs in the homeostasis of zinc and copper has been attributed to the β-domain. Recently, it has been shown that an oxidoreductive mechanism modulates the affinity of zinc for the cysteine thiolate ligands and that key players are reduced glutathione, oxidized glutathione, and other oxidizing agents. In this regard, cysteines of metal thiolate clusters confer redox sensitivity to an otherwise redox inert metal ligand (e.g., Zn 2+ ) and facilitate the potential of MTs to participate in intracellular signal transduction pathways [START_REF] Jacob | Control of zinc transfer between thionein, metallothionein, and zinc proteins[END_REF][START_REF] Jiang | The glutathione redox couple modulates zinc transfer from metallothionein to zinc-depleted sorbitol dehydrogenase[END_REF][START_REF] Maret | Metallothionein/disulfide interactions, oxidative stress, and the mobilization of cellular zinc[END_REF][START_REF] Maret | Thiolate ligands in metallothionein confer redox activity on zinc clusters[END_REF]. In this respect, the observation of NO-induced selective release of bound metals from MTs points to a possible physiological function of this interaction. In particular, MTs act as scavengers of free NO by forming S-nitrosothiols with a concomitant release of zinc from the β -domain [START_REF] Zangger | Nitric oxide selectively releases metals from the amino-terminal domain of metallothioneins: potential role at inflammatory sites[END_REF].

The question now is whether the S-nitrosylable Cys residues of MTs may be highly reactive to S-glutathionylation as well. In this study, S-glutathionylation of MTs under nitrosative/oxidative stress has been addressed by exposing the MT to three different oxidants (i.e., GSNO, diamide and H 2 O 2 ) acting by different mechanisms. GSNO is the S-nitrosothiol of GSH and it could be the most relevant biological molecule to carry out nitrosation reactions under physiological conditions [START_REF] Arnelle | NO+, NO, and NO-donation by S-nitrosothiols: implications for regulation of physiological functions by S-nitrosylation and acceleration of disulfide formation[END_REF][START_REF] Meyer | Kinetics and equilibria of Snitrosothiol-thiol exchange between glutathione, cysteine, penicillamines and serum albumin[END_REF][START_REF] Padgett | S-nitrosoglutathione reversibly inhibits GAPDH by Snitrosylation[END_REF]. Diamide, which is a strong oxidant with high specificity for thiols [START_REF] Kosower | Diamide: an oxidant probe for thiols[END_REF], and H 2 O 2 , a physiologically occurring oxidant, all have been shown to cause rapid, extensive, and reversible S-glutathionylation of a number of cell proteins [START_REF] Dalle-Donne | Sglutathionylation in human platelets by a thiol-disulfide exchange-independent mechanism[END_REF][START_REF] Fratelli | Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes[END_REF][START_REF] Ghezzi | Protein glutathionylation: coupling and uncoupling of glutathione to protein thiol groups in lymphocytes under oxidative stress and HIV infection[END_REF][START_REF] Kosower | Diamide: an oxidant probe for thiols[END_REF][START_REF] Rossi | Oxidized forms of glutathione in peripheral blood as biomarkers of oxidative stress[END_REF]. Both Western blot and dot blot analyses have unequivocally shown that MT can be glutayhionylated in vitro. Moreover, it appears that glutathionylation is accompanied by polimerization of MT, suggesting that modification of Cys residues be one of the structural determinants at the basis of the well known tendency of MT to aggregate.

The immunochemical evidence for S-glutathionylation of MT in lysate samples was obtained by Western blot, using monoclonal antibodies raised to GSH as reported elsewhere [START_REF] Dalle-Donne | Reversible Sglutathionylation of Cys 374 regulates actin filament formation by inducing structural changes in the actin molecule[END_REF][START_REF] Dalle-Donne | Sglutathionylation in human platelets by a thiol-disulfide exchange-independent mechanism[END_REF][START_REF] Kil | Regulation of mitochondrial NADP+-dependent isocitrate dehydrogenase activity by glutathionylation[END_REF][START_REF] Pastore | Actin glutathionylation increases in fibroblasts of patients with Friedreich' s ataxia: a potential role in the pathogenesis of the disease[END_REF][START_REF] Sparaco | Protein glutathionylation in human central nervous system: potential role in redox regulation of neuronal defense against free radicals[END_REF]. It is interesting to note that MT was found to be highly aggregated in vivo, yet heavily glutathionylated. In this respect, while our data refer to in vitro conditions that most likely do not occur as such in vivo, nevertheless they constitute a valid model of physiological thiol modifications, unequivocally showing a link between cysteines modification and polymers formation.

The possibility that the same Cys residues sensitive to S-nitrosylation may be reactive for S-glutathionylation has been evaluated by analyzing the structural aspects and the molecular details of the oxidative protein modification. Analysis of the three-dimensional structure of rat liver MT evidences that Cys residues of the N-terminal domain, given their solvent accessibility, are the likely targets of S-glutathionylation, taking also into account the higher bulkiness of glutathione as compared to NO. Further, the observation that two Cys residues of MT N-terminal domain have basic residues in their immediate neighborhood indicates that these residues can be more reactive, especially in the case that partial apo forms of MT are present.

From this viewpoint, S-glutathionylation of MT, given its reversibility, would provide protection from irreversible oxidation of Cys residues, thus representing a mechanism of high potential biological relevance. In fact, while S-nitrosylation does not prevent further and irreversible oxidation of Cys residues, S-glutathionylation can be reversed provided the GSH/GSSG ratio is high or, in other words, in conditions in which the oxidative stress within the cell has not reached a critical level. This process would regenerate the free thiol group of Cys residues thus restoring the metal binding ability and hence the biological function of MT. [START_REF] Pettersen | UCSF Chimera--a visualization system for exploratory research and analysis[END_REF].

FIGURE LEGENDS

Fig. 1 .

 1 Fig. 1. Western blot and dot blot analyses of S-glutathionylation of MT induced by GSNO

Fig. 2 .

 2 Fig. 2. Dot blot analysis of S-glutathionylation of MT induced by treatment with diamide and

Fig. 3 .

 3 Fig. 3. Western blot analysis of S-glutathionylation of MT in peripheral blood mononuclear

Fig. 4 .

 4 Fig. 4. Schematic representation of the three-dimensional structure of rat MT. For clarity only

Fig. 4

 4 Fig.4

Fig.1

Fig.2

Fig.3

Acknowledgements

This study was supported by grants from EU Commission (Project ZINCAGE, n.

FOOD- CT-2003-506850) to M. Colasanti.


Kai-Christian Müller
email: kc.mueller@pulmoresearch.de

Kirsten Paasch
email: k.paasch@pulmoresearch.de

Beke Feindt
email: b.feindt@pulmoresearch.de

Lutz Welker
email: l.welker@gmx.net

Henrik Watz
email: h.watz@pulmoresearch.de

Michael Weise
email: m_weise@pneumo-lindenhof.ch

Ralph A Schmid
email: ralph.schmid@insel.ch

Masaki Nakashima
email: m.nakashima@kh-grosshansdorf.de

Detlev

Detlev Branscheid
email: d.branscheid@kh-grosshansdorf.de

Helgo Magnussen
email: magnussen@pulmoresearch.de

Rudolf A Jörres
email: rudolf.joerres@med.uni-muenchen.de

Olaf Holz
email: o.holz@pulmoresearch.de

In contrast to lung

Keywords: ageing, beta-galactosidase, COPD, fibroblasts, IGFBP, lung emphysema, proliferation, senescence

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

A C C E P T E D M A N U S C R I P T

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Abstract

Smoking is known to be linked to skin ageing and there is evidence for premature senescence of parenchymal lung fibroblasts in emphysema. To reveal whether the emphysema-related changes in cellular phenotype extend beyond the lung, we compared the proliferation characteristics of lung and skin fibroblasts between patients with and without emphysema. Parenchymal lung fibroblasts and skin fibroblasts from the upper torso (thus limiting sun exposure bias) were obtained from patients without, or with mild, or with moderate to severe emphysema undergoing lung surgery.

We analysed proliferation rate, population doublings (PD), staining for senescence-associated beta-galactosidase (β-gal) and gene expression of IGFBP-3 and IGFBP-rP1. Population doubling time of lung fibroblasts differed between control, mild, and moderate to severe emphysema (median (IQR) 29.7(10.0), 33.4(6.1), 44.4(21.2) h; p=0.012) and staining for β-gal was elevated in moderate to severe emphysema. Compared to control subjects, skin fibroblasts from patients with emphysema did not differ with respect to proliferation rate, PD and β-gal staining, and showed a lower abundance of mRNA for each). These results suggest that the induction of a senescent fibroblast phenotype by cigarette smoke, as observed in emphysema, primarily occurs in the lung.

Introduction

The destruction of alveolar walls and resultant reduction of elastic recoil are hallmarks of lung emphysema [START_REF] Gold | Global Initative for Chronic Obstructive Lung Disease. Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease[END_REF]. Fibroblasts provide an important part of the lung's structural support and extracellular matrix that is essential for its integrity [START_REF] Absher | Fibroblasts[END_REF]. Based on clinical and experimental data it has been hypothesized that the long-term exposure to noxious agents or cigarette smoke, the major cause for emphysema, could result in premature ageing of lung fibroblasts [START_REF] Holz | Lung fibroblasts from patients with emphysema show a reduced proliferation rate in culture[END_REF][START_REF] Nyunoya | Cigarette smoke induces cellular senescence[END_REF]. Such a change in fibroblast phenotype could be responsible for an impaired structural maintenance of the lung and could thereby contribute to the development of emphysema [START_REF] Nyunoya | Cigarette smoke induces cellular senescence[END_REF]Tuder et al. 2006).

In line with this, lung fibroblasts from patients with emphysema exhibit a reduced proliferation rate, lower number of population doublings in serial passaging [START_REF] Holz | Lung fibroblasts from patients with emphysema show a reduced proliferation rate in culture[END_REF], and altered growth factor response [START_REF] Holz | Lung fibroblasts from patients with emphysema show a reduced proliferation rate in culture[END_REF][START_REF] Müller | Different proliferative capacity of lung fibroblasts obtained from control subjects and patients with emphysema[END_REF]. In addition, we found an increased staining for senescence-associated beta-galactosidase (β-gal, [START_REF] Dimri | A biomarker that identifies senescent human cells in culture and in aging skin in vivo[END_REF]), as well as increased mRNA expression of the insulin-like growth factor-binding proteins (IGFBP)-3 and -rP1 in these cells [START_REF] Müller | Different proliferative capacity of lung fibroblasts obtained from control subjects and patients with emphysema[END_REF]). Both proteins have been shown earlier to be senescenceassociated [START_REF] Goldstein | Insulin-like growth factor binding protein 3 accumulates to high levels in culture medium of senescent and quiescent human fibroblasts[END_REF]Swisshelm et al. 1995;Wilson et al. 2002).

As a clinical sign of ageing, the extent of skin wrinkling has been evaluated and, in fact, shown to be associated with emphysema [START_REF] Patel | Smoking related COPD and facial wrinkling: is there a common susceptibility?[END_REF]. Skin wrinkling ranges among smokinginduced alterations which also comprise a rise in the proportion of elastic fibres [START_REF] Frances | Changes in the elastic tissue of the non-sun-exposed skin of cigarette smokers[END_REF][START_REF] Just | Effect of smoking on skin elastic fibres: morphometric and immunohistochemical analysis[END_REF]. Noteworthy enough, this increase was found to be associated with lung function impairment, suggesting a common effect of smoking on elastic fibres in both lung and skin [START_REF] Just | Relationships between lung function, smoking and morphology of dermal elastic fibres[END_REF]. This might be linked to disturbances in protease/anti-protease balance, as elevated MMP-1 gene expression has been observed in skin biopsies of active smokers compared to non-smokers [START_REF] Lahmann | Matrix metalloproteinase-1 and skin ageing in smokers[END_REF]. Moreover, in vitro experiments revealed that cigarette smoke extract can raise the expression of MMP-1 and -3 in skin fibroblasts of healthy individuals (Yin et al. 2000). As fibroblasts are basically involved in the maintenance of the extracellular matrix [START_REF] Absher | Fibroblasts[END_REF], alterations of this function, as observed in ageing, could well be associated with an altered, particularly aged, cellular phenotype.

In the present study we therefore aimed to reveal, whether skin fibroblasts of patients with emphysema showed evidence for cellular senescence in terms of proliferation rate and capacity, as well as staining for β-gal and gene expression of IGFBPs. For this purpose we compared parenchymal lung and skin fibroblasts within individuals in patients with and without lung emphysema.

A C C E P T E D M A N U S C R I P T

-4 -

Material and Methods

Patients

The study population (

Fibroblasts

Lung fibroblasts were derived from surgical tissue [START_REF] Holz | Lung fibroblasts from patients with emphysema show a reduced proliferation rate in culture[END_REF]). Briefly, the macroscopic appearance of the specimen was examined and pleura-free parenchymal samples were taken from non-cancerous areas. Samples were washed, minced (1-2 mm 2 ) and transferred into 25 cm 2 culture dishes (Sarstedt, Nürnbrecht, Germany) for primary explant culture in Dulbecco's modified Eagle's medium (DMEM) containing 10 % foetal calf serum (FCS, same batch used throughout the study), 100 U/mL Penicillin, 100 µg/mL Streptomycin, and 50 µg/mL Gentamycin (all cell culture reagents: Invitrogen). Following primary culture, cells were trypsinized, resuspended in culture medium plus 10 % dimethylsulfoxide (DMSO), stored for 24 h at -80°C and transferred into liquid nitrogen. Skin samples of size 2-8 mm 2 were taken during surgery from the wound edge located at the patients' upper torso. The tissue was minced, immediately transferred to explant culture and treated as described for lung fibroblasts.

Assessment of proliferation

Cell proliferation and serial passaging were performed as previously described [START_REF] Holz | Lung fibroblasts from patients with emphysema show a reduced proliferation rate in culture[END_REF]). Proliferation rate was assessed in passage 2 (about 3-4 population doublings after primary culture) in 24 well-dishes. Cell number was determined in triplicate after 24, 48, 72, and 96 h.

Mean cell numbers were logarithmically transformed and the linear regression slope was calculated to derive the doubling time (DT). For serial passaging, cells were thawed and cultured

A C C E P T E D M A N U S C R I P T

-5 -for about 5 days before transfer to passage 2. Cells were seeded at a density of 100 000 cells per 25 cm 2 , grown for one week, harvested, and seeded again. When the number of harvested cells dropped below the seeded number, the culture was terminated and the cumulative number of population doublings (PD) calculated.

Staining for senescence-associated beta-galactosidase (β-gal)

Following a previously described procedure [START_REF] Müller | Different proliferative capacity of lung fibroblasts obtained from control subjects and patients with emphysema[END_REF], 10 000 -15 000 fibroblasts were incubated for 24 h on glass slides. Slides were then stained for β-gal activity according to the manufacturer's instructions (Cell Signaling Technologies, Beverly, MA, USA). The analysis was performed in cells of passage 4 or 5 after primary culture. Cells positive for the blue stain were counted under visible light, while counter-staining with DAPI enabled the unambiguous determination of cell numbers under UV light.

Gene expression analysis

The abundance of mRNA for IGFBPs was analysed by qPCR using the Lightcycler Instrument (Roche, Mannheim, Germany). Cells were thawed, cultured up to passage 3, harvested and stored frozen until RNA isolation and cDNA transcription. Primers [START_REF] Müller | Different proliferative capacity of lung fibroblasts obtained from control subjects and patients with emphysema[END_REF]) and protocols [START_REF] Erpenbeck | Segmental allergen challenge in patients with atopic asthma leads to increased IL-9 expression in bronchoalveolar lavage fluid lymphocytes[END_REF] were those used previously. Abundance of mRNA was determined via the ratio of target to house-keeping gene (PBGD, porphobilinogen deaminase).

Data Analysis

Owing to skewed data distributions in some variables, median values and quartiles or interquartile ranges (IQR) were chosen for all variables to achieve a homogeneous description. For non-normally distributed variables the Kruskal-Wallis test was employed for the comparison of groups and the Mann-Whitney U-test for specific post hoc comparisons. The Wilcoxon test was used for paired comparisons. Additionally, analysis of variance (ANOVA) was performed on logtransformed data of non-normally distributed variables to utilise the power of post hoc comparisons by the Newman-Keuls-statistics. Correlation analysis was performed by computation of linear correlation coefficients. P-values <0.05 were considered statistically significant.

Results

In both skin and parenchymal lung fibroblasts proliferation rates were determined in 18 control patients, 13 patients with mild, and 11 patients with moderate to severe emphysema. (Table 1A).

The doubling time of lung fibroblasts differed between the three groups analysed (ANOVA, 1A) with the longest mean doubling time in patients with moderate to severe emphysema. No such difference was detected in skin fibroblasts of the same patients (ANOVA, p=0.54; Figure 1B). In control patients the doubling time of skin fibroblasts was higher compared to lung fibroblasts (t-test, p=0.005), while there was no such difference in the emphysema groups.

A C C E P T E D M A N U S C R I P T -6 - p=0.012, Figure
The mean (95%-CI) ratio between doubling times of lung relative to skin fibroblasts was 84.8 (73.2; 96.4) % in control subjects, 100.9 (85.0; 116.8) % in mild emphysema, and 130.3 (85.0; 175.5) % in moderate to severe emphysema (ANOVA, p=0.02).

Neither skin nor lung fibroblasts showed a correlation between doubling time and the donors' age or number of packyears, neither in the separate groups nor the total group. The correlation between doubling times of skin and lung fibroblasts was significant in control (r=0.50, p=0.04),

but not in mild (r=-0.10, p=0.74) or moderate to severe emphysema (r=-0.19, p=0.57).

As the group of patients with mild emphysema did not show a pronounced difference in doubling time compared to control patients, the subsequent experiments were performed with all available fibroblasts strains from patients with moderate-severe emphysema and randomly selected fibroblast strains from a matched number of control patients (Table 1B).

Cumulative population doublings (PD) of skin fibroblasts were assessed until the number of harvested cells fell below the number of seeded cells. The median (quartiles) number of PD in control patients was 8.3 (7.4; 9.0), in emphysema 8.1 (5.9; 13.0). These values were not significantly different from each other.

In control patients the median (quartiles) percentage of β-gal positive skin fibroblasts was 2.9

(1.9; 3.4) %, in emphysema 4.9 (3.1; 5.7) % (p=0.12). The corresponding lung fibroblasts showed percentages of 5.4 (4.3; 6.8) % in control patients, and of 17.0 (13.0; 20.9) % in emphysema (p=0.012). In both groups the proportion of cells staining for β-gal was lower in skin compared to lung fibroblasts (p<0.05, each). Doubling time and β-gal staining were not correlated with each other, neither in skin nor in lung.

In skin fibroblasts of patients with emphysema the median (quartiles) relative abundance of mRNA for IGFBP-3/PBGD and for IGFBP-rP1/PBGD was lower (Mann-Whitney U-test, p=0.04, p=0.02) than in control patients (Figure 2). In lung fibroblasts, the relative abundance of mRNA for IGFBP-3/PBGD and for IGFBP-rP1/PBGD was higher (Figure 2). Taken overall, there were significant correlations between the expression of IGFBP-3 and IGFBP-rP1 in skin fibroblasts (r=0.56, p=0.03) and in lung fibroblasts (r=0.75, p=0.003). The ratio of normalised mRNA abundance of skin to lung fibroblasts for IGFBP-3 was 1.9 (1.7; 2.3) in control patients and 1.3

(1.1; 1.5) in severe emphysema (p=0.014). The corresponding ratios were lower for IGFBP-rP1

(1.1 (1.0; 1.9) vs. 1.0 (0.8; 1.1)) and not significantly different (p=0.26).

A C C E P T E D M A N U S C R I P T

-7 -

Discussion

In the present study we compared the in vitro proliferation of lung and skin fibroblasts from the same individuals between patients with and without emphysema. Parenchymal lung fibroblasts showed a reduced proliferation rate in moderate to severe emphysema, which is in line with our previous findings [START_REF] Holz | Lung fibroblasts from patients with emphysema show a reduced proliferation rate in culture[END_REF]). There was, however, no such difference for skin fibroblasts.

The proportion of skin fibroblasts positive for senescence-associated beta-galactosidase was similar between groups and lower than in lung fibroblasts. The abundance of mRNA for senescence-associated IGFBP-3 or -rP1, was lower in skin fibroblasts of emphysema compared to control patients. These findings suggest that smoking-related changes in fibroblast phenotype as found in emphysema occur primarily in the lung (Table 2).

Smoking has long been known to affect skin appearance, in particular by promoting facial skin wrinkling [START_REF] Daniell | Smoker's wrinkles. A study in the epidemiology of "crow's feet[END_REF][START_REF] Kadunce | Cigarette smoking: risk factor for premature facial wrinkling[END_REF][START_REF] Koh | Cigarette smoking associated with premature facial wrinkling: image analysis of facial skin replicas[END_REF][START_REF] Patel | Smoking related COPD and facial wrinkling: is there a common susceptibility?[END_REF]). The effect is enhanced by sunlight (Yin et al. 2001), but alterations of elastic tissue properties also occur in sunprotected areas [START_REF] Frances | Changes in the elastic tissue of the non-sun-exposed skin of cigarette smokers[END_REF][START_REF] Helfrich | Effect of smoking on aging of photoprotected skin: evidence gathered using a new photonumeric scale[END_REF]). Accordingly, MMP-1 which can act on elastic fibres has been found up-regulated in the buttock skin of smokers [START_REF] Lahmann | Matrix metalloproteinase-1 and skin ageing in smokers[END_REF].

However, the effect of smoking on skin wrinkling is not unopposed [START_REF] Malvy | Epidemiologic determinants of skin photoaging: baseline data of the SU.VI.MAX. cohort[END_REF][START_REF] O'hare | Tobacco smoking contributes little to facial wrinkling[END_REF], and in some cases the effect of UV light may outweigh that of smoking. Both smoking and UV-light induce changes in the morphology of elastic fibres [START_REF] Frances | Changes in the elastic tissue of the non-sun-exposed skin of cigarette smokers[END_REF][START_REF] Just | Effect of smoking on skin elastic fibres: morphometric and immunohistochemical analysis[END_REF]). Remarkably, increased amounts and morphological abnormalities of elastic fibres in the reticular dermis of the skin were found to be associated with lung function impairment [START_REF] Just | Relationships between lung function, smoking and morphology of dermal elastic fibres[END_REF]. Thus there might be a common effect of smoking on tissue properties of both lung and skin.

Fibroblasts could be involved in the increased MMP-1 gene expression, as in vitro experiments revealed that cigarette smoke extract increased the expression of MMP-1 and -3 in skin fibroblasts of healthy individuals (Yin et al. 2000). Moreover, it blocked the cells' responsiveness to TGF-β1 (Yin et al. 2003). As TGF-β directly stimulates fibroblasts to produce extracellular matrix and collagen, this could mediate the observed lower collagen production in smokers [START_REF] Jorgensen | Less collagen production in smokers[END_REF]. Interestingly, lung fibroblasts of patients with emphysema exhibit reduced responsiveness to exogenous TGF-β1 (Togo S. et al. unpublished data).

While lung fibroblasts are probably more or less directly affected by smoking, cells of other body compartments might be exposed to tobacco smoke compounds or metabolites carried by the bloodstream. Water-soluble compounds of cigarette smoke exert effects on lung fibroblasts [START_REF] Carnevali | Cigarette smoke extract inhibits fibroblast-mediated collagen gel contraction[END_REF][START_REF] Rocca | Cigarette smoke exposure inhibits extracellular MMP-2 (gelatinase A) activity in human lung fibroblasts[END_REF]) and these effects can be lasting, apparently altering their phenotype [START_REF] Nyunoya | Cigarette smoke induces cellular senescence[END_REF]). Based on this, the comparison of fibroblasts from lung and skin of the same individuals seemed reasonable to address the question whether smoking exerts

A C C E P T E D M A N U S C R I P T

-8 -parallel effects in different compartments and induces a common fibroblast phenotype.

However, the skin fibroblasts studied exhibited similar proliferation characteristics in control and emphysema patients. This is unlikely to have been the result of differential cell culture bias, as the lung fibroblasts showed a difference, confirming our previous findings [START_REF] Holz | Lung fibroblasts from patients with emphysema show a reduced proliferation rate in culture[END_REF]) in newly recruited patients (only lung fibroblasts of four patients were included in our previous study [START_REF] Holz | Lung fibroblasts from patients with emphysema show a reduced proliferation rate in culture[END_REF]). It has also been shown previously, that the reduced proliferation rate in lung fibroblasts of patients with emphysema is not associated with an increase in apoptosis or necrosis [START_REF] Holz | Lung fibroblasts from patients with emphysema show a reduced proliferation rate in culture[END_REF]). The fact that we did not observe a relationship between donor age and proliferation rate is compatible with data from other studies [START_REF] Cristofalo | Relationship between donor age and the replicative lifespan of human cells in culture: a reevaluation[END_REF].

Foetal skin fibroblasts appear to show a lower proliferation rate than lung fibroblasts [START_REF] Schneider | Tissue-specific differences in cultured human diploid fibroblasts[END_REF]. In line with this, the ratio of doubling times between lung and skin was <1 in control patients, whereas in emphysema the slow proliferation of lung fibroblasts raised this ratio to values >1. Skin fibroblasts of both groups did not differ in their number of total population doublings.

Compared to lung fibroblasts [START_REF] Holz | Lung fibroblasts from patients with emphysema show a reduced proliferation rate in culture[END_REF]), values were lower in skin fibroblasts, in line with previous findings [START_REF] Schneider | Tissue-specific differences in cultured human diploid fibroblasts[END_REF]. Moreover, the proportion of skin fibroblasts positive for β-gal was not significantly elevated in emphysema. Levels of staining were much lower than in parenchymal lung fibroblasts. In accordance with our previous results [START_REF] Müller | Different proliferative capacity of lung fibroblasts obtained from control subjects and patients with emphysema[END_REF], staining in lung fibroblasts was more pronounced in patients with moderate to severe emphysema as compared to the control group, suggesting that a senescent phenotype is limited to lung fibroblasts in patients with emphysema. Both doubling time and β-gal staining were significantly increased in emphysema but did not correlate with each other. Although in general replicative age correlates with β-gal staining [START_REF] Dimri | A biomarker that identifies senescent human cells in culture and in aging skin in vivo[END_REF], the interindividual variability of both markers was likely to be responsible for a lack of correlation in our study. In a previous study we had observed such a correlation within serial passages of the same strains of fibroblasts [START_REF] Müller | Different proliferative capacity of lung fibroblasts obtained from control subjects and patients with emphysema[END_REF].

IGFBP-3 and -rP1 can indicate cellular senescence [START_REF] Goldstein | Insulin-like growth factor binding protein 3 accumulates to high levels in culture medium of senescent and quiescent human fibroblasts[END_REF]Swisshelm et al. 1995;Wilson et al. 2002) and their mRNA abundance has been shown to be elevated in lung fibroblasts of emphysema [START_REF] Müller | Different proliferative capacity of lung fibroblasts obtained from control subjects and patients with emphysema[END_REF]. Skin fibroblasts of control patients showed a relative mRNA abundance of IGFBP-3 about two-fold higher than lung fibroblasts. This might explain their lower proliferation as compared to lung fibroblasts, as IGFBP-3 is known to be a proliferation inhibitor [START_REF] Oh | Insulin-like growth factor (IGF)independent action of IGF-binding protein-3 in Hs578T human breast cancer cells. Cell surface binding and growth inhibition[END_REF]. In emphysema this ratio was close to one, due to lower levels in skin and higher levels in the lung. Abundance of mRNA for IGFBP-rP1 was similar in skin and lung fibroblasts. As two control patients showed exceptional values, the difference between groups regarding lung fibroblasts was less pronounced than found previously [START_REF] Müller | Different proliferative capacity of lung fibroblasts obtained from control subjects and patients with emphysema[END_REF]). Taking into account the similar proliferation characteristics of skin, in contrast to lung,

A C C E P T E D M A N U S C R I P T -9 -
fibroblasts in the groups, the expression patterns of IGFBP-3 and -rP1 could not be considered as unequivocal markers of cellular senescence.

The recent observations [START_REF] Patel | Smoking related COPD and facial wrinkling: is there a common susceptibility?[END_REF]) on a correlation between emphysema and facial wrinkling are of great interest with regard to the factors involved. Facial skin is regularly exposed to UV-light, whereas the skin fibroblasts used in the present study were not, as they were derived from the upper torso. Probably induced senescence requires an appropriate, sufficient exposure, which might be cigarette smoke in the lung or UV-light in the skin. Moreover, effects by smoking or UV-light comprise alterations of elastic fibres. MMP up-regulation in smokers [START_REF] Lahmann | Matrix metalloproteinase-1 and skin ageing in smokers[END_REF] might represent an acute effect in vivo, in parallel to acute in vitro effects of cigarette smoke extract (Yin et al. 2000). However, skin fibroblast proliferation was assessed after at least 4 weeks in culture, thus acute effects of smoking-related compounds were excluded. The proliferation data did not indicate persistent alterations in skin fibroblast phenotype, in contrast to lung fibroblast data as reported here and previously [START_REF] Holz | Lung fibroblasts from patients with emphysema show a reduced proliferation rate in culture[END_REF][START_REF] Müller | Different proliferative capacity of lung fibroblasts obtained from control subjects and patients with emphysema[END_REF]). The most plausible explanation is that the induction of acute versus persistent changes in cell function depends on the strength of the exposure, and that persistent effects, especially those on proliferation behaviour, need higher local doses than temporary effects on cell function.

Taken together, the findings of our study indicate that fibroblast phenotype was not grossly altered in samples of sun-protected skin of patients with lung emphysema compared to control smokers. This suggests that smoking induced cellular senescence is primarily found in the lung. Photodermatol.Photoimmunol.Photomed. 17, 178-183. Yin, L., Morita, A., Tsuji, T., 2003. Tobacco smoke extract induces age-related changes due to modulation of TGF-beta. Exp.Dermatol. 12 Suppl 2, 51-56.

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-16 -

Future

  Figure 1 -Fibroblast proliferation Population doubling time (DT) of fibroblasts (h); open circles represent single data points for individual patients, closed circles and whiskers represent geometric mean values and SEM for each group. (A) lung fibroblasts (ANOVA, p=0.012), (B) skin fibroblasts (ANOVA, p=0.54).

Figure 2 -

 2 Figure 2 -Relative mRNA abundance of IGFBP-3 and -rP1 Relative mRNA abundance of IGFBP-3 and IGFBP-rP1 each in relation to PBGD. Circles / whiskers represent the respective group median values / quartiles; open circles: fibroblasts from patients with moderate to severe emphysema; closed circles: fibroblasts from control patients.

  Fig.1

Table 1

 1

	) comprised 18 control patients (C) who did not show signs of
	emphysema, 13 patients with mild emphysema (ME), and 11 patients with moderate to severe
	emphysema (E). Patients were undergoing surgery for lung tumour resection, except two who had
	lung volume reduction surgery. Patients were assigned to their respective group by two
	independent investigators (RAJ, HW) who were blinded with respect to the in vitro results and
	took into account all available information, including the patient's history, symptoms, lung
	function, chest X-ray/CT, bronchoscopic, and histological findings. When evaluating expiratory
	flow-volume curves and body plethysmographic resistance loops, special attention was paid to
	their characteristic form in emphysema, as well as to lung hyperinflation. In addition, data on
	single-breath diffusing capacity for carbon monoxide were available in a number of patients (9 C/
	12 ME/ 6 E). The study was approved by the local Ethics Committee and all patients gave their
	written informed consent.

Table 1 -

 1 Patient characteristicsA) Patient characteristics (all patients): The table shows median values and quartiles (in parentheses). ANOVA, Newman-Keuls-post-hoc analysis: *p<0.05, **p<0.01: control vs. mild

	or severe emphysema. + p<0.05, ++ p<0.01: mild vs. severe emphysema. BMI = body mass
	index, VC = vital capacity, FEV 1 = forced expiratory volume in one second, ITGV = intra-
	thoracic gas volume, RV = residual volume, TLC = total lung capacity, yr = years, 1 reliable
	data available in 14 control subjects, 7 mild emphysema and 5 moderate-to-severe emphysema
	patients; all patients were current or ex-smokers except for one never-smoking control patient.
	Predicted values were taken form ERS guidelines (Quanjer et al. 1993), stages of COPD
	according to GOLD guidelines (GOLD 2006). 2 n.a. = not applicable for patients with
	FEV 1 /FVC>70% but FEV 1 <80% as lung function impairment was most likely due to lung
	tumour. 3 One subject was included who was functionally not limited in terms of GOLD
	criteria but showed clear macroscopic evidence for emphysematous alterations in the resected
	lung tissue.

B) Patient characteristics (subgroups): Main characteristics and case numbers of patients subgroups included in the analysis of the respective markers are stated here. Age and FEV 1 are displayed as median (IQR); for all markers analysed these subgroups did not differ in age, BMI or smoking history.

Table 2 -Alterations in fibroblasts from patients with emphysema

 2 The table summarises the results obtained in different studies on cultures of primary fibroblasts from patients with emphysema. The effects are presented as relative changes as compared to fibroblasts from control patients without emphysema. The repair capacity model comprises the ability to contract collagen gels and the extend of migration towards different stimuli. Swisshelm, K.,Ryan, K., Tsuchiya, K., Sager, R., 1995. Enhanced expression of an insulin growth factor-like binding protein (mac25) in senescent human mammary epithelial cells and induced expression with retinoic acid. Proc.Natl.Acad.Sci.U.S.A. 92, 4472-4476. Tuder, R.M., Yoshida, T., Arap, W., Pasqualini, R., Petrache, I., 2006. State of the art. Cellular and molecular mechanisms of alveolar destruction in emphysema: an evolutionary perspective. Proc.Am Thorac.Soc. 3, 503-510. Wilson, H.M., Birnbaum, R.S., Poot, M., Quinn, L.S., Swisshelm, K., 2002. Insulin-like growth factor binding protein-related 1 inhibits proliferation of MCF-7 breast cancer cells via a senescence-like mechanism. Cell Growth Differ. 13, 205-213. Yin, L., Morita, A., Tsuji, T., 2000. Alterations of extracellular matrix induced by tobacco smoke extract. Arch.Dermatol.Res. 292, 188-194. Yin, L., Morita, A., Tsuji, T., 2001. Skin aging induced by ultraviolet exposure and tobacco

	1 Holz et al. 2004; 2 S. Togo, unpublished data; n.d.: not determined

Table 1 A) Patient characteristics (all patients)

 1

			Control		Mild	Moderate to severe
					emphysema	emphysema
	n		18		13	11
	Sex	m / f	14 / 4		9 / 4	10 / 1
	Age	yr	65 (58; 73)	66 (61; 72)	62	(53; 71)
	BMI	kg/m 2	25.4 (22.9; 27.6)	23.5 (21.1; 27.8)	22.8	(17.7; 26.3)
	VC	%pred	90 (81; 108)	99 (87; 118)	89	(72; 106)
	FEV 1	%pred	80 (75; 101)	73 (65; 83)	48	(34; 63) ** ,++
	FEV 1 /VC %pred	96 (82; 102)	82 (74; 83) *	54	(41; 68) ** ,++
	ITGV	%pred	99 (85; 122)	124 (114; 156) *	182	(155; 219) ** ,++
	RV	L	2.48 (2.23; 2.60)	2.92 (2.26; 3.27)	5.00	(4.00; 6.13) ** ,++
	TLC	L	5.59 (5.24; 6.39)	6.26 (5.37; 6.72)	8.60	(7.24; 10.15) ** ,++
	RV/TLC	%	41 (36; 45)	48 (42; 53) *	58	(50; 70) ** ,+
	Packyr 1		45 (30; 52)	45 (15; 75)	40	(30; 40)
	GOLD -/I/II/III/VI/ 2 9/0/4/0/0/5		2/3/6/0/0/2	1 3 /0/3/7/0/0
	B) Patient characteristics (subgroups)
				N	Age (yr)	FEV 1 (%pred)
	Serial Passaging	C	9	59 (18)	85 (19)
			E	8	62 (15)	37 (24) *
	beta-Galactosidase	C	9	61 (7)	88 (19)
			E	9	63 (11)	37 (29) *
	Gene expression	C	8	59 (15)	91 (25)
			E	7	58 (14)	36 (29)*

Table 2 :

 2

		lung fibroblasts	skin fibroblasts
	Population doubling time
	Serial passaging (cumPD)	1
	β-gal staining
	Repair capacity	2	n.d.
	IGFBP-3 , IGFBP-rP1
	PGE2	2	n.d.

Acknowledgements

We would like to thank all patients for their co-operation and support of our research in a situation in which, conceivably, their major concern was lung surgery as a life-determining event.

Furthermore we would like to thank all colleagues from the Department of Thoracic Surgery, Hospital Grosshansdorf, especially Dr. Albrecht, Dr. Diemel, Dr. Halberstadt and Dr. Neuhauss for their support in this study.

Disclosure Statement

The authors declare that they have no competing interests. The interpretation and presentation of these results do not influence the personal or financial relationship of any of the authors with other people or organisations.

The Role of your Funding Source

The study was funded by Deutsche Rentenversicherung Nord. The funding source had no influence in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.