Rapha Lle Varraso
email: <raphaelle.varraso@inserm.fr

Nicole Massin

Michel Hery

Martine Fradier-Dusch

Jean-Pierre Michaely

Maryvonne Fournier

Genevi Ve Hubert

Patrick Biette

Benoit Rieger

Aline Berthelin

G Rard Hecht

Rachel Nadif

Epid Miologie

Not only training but also exposure to chlorinated compounds generates a response to oxidative stimuli in swimmers

Keywords: Adolescent, Adult, Antioxidants, pharmacology, Asthma, etiology, Chlorine Compounds, poisoning, Data Collection, Humans, Male, Multivariate Analysis, Oxidative Stress, Physical Endurance, Respiratory Tract Diseases, etiology, Risk Assessment, Swimming, physiology biological markers, oxidative stress, swimming, chloramine, trihalomethanes, physical exercise. reactions

Mechanism of toxicity of chlorinated compounds on health were not totally identified and oxidant pathway has been proposed as mediators of their toxicity. Relations between exposure to chlorinated compounds and biological markers of response to oxidative stimuli were investigated in swimmers, taking into account the effect of training. Twenty-two male swimmers aged 15 25 years have been surveyed twice. Prevalence of irritant symptoms and asthma, and number of hours of training were reported. Exposure to NCl and blood response to oxidative stimuli (catalase, superoxide dismutase, 3 glutathione peroxidase activities and ceruloplasmin, ferritin and total antioxidant concentrations) were measured. Univariate analysis were completed by multivariate analysis. High prevalences of irritant symptoms and asthma were found. Multivariate analysis confirmed the results of univariate analyses and showed that Cu /Zn SOD activity was increased by exposure and by training (p 0.01, p 0.0001 respectively). Erythrocyte ++ ++ = = GSH-Px was decreased whereas plasma GSH-Px was increased by exposure (p 0.002, p 0.002). No other association was found. = = Higher irritant symptoms and increases in the activities of erythrocyte Cu /Zn SOD and of plasma GSH-Px with exposure ++ ++ support well the hypothesis that the production of reactive oxygen species is not only related to training but also to exposure to chlorinated compounds.

Introduction

The effects of chlorinated compounds on health and the mechanism of their toxicity were not totally and clearly identified, even if chlorination is the oldest and the most widely used method of water disinfection. The use of chlorine-containing agents for the disinfection of water in swimming pool leaded to the formation of various chlorinated compounds, chloramines and halomethanes, resulting from

). The continuous production of ROS may overwhelmed antioxidant defenses and result in an oxidative stress [START_REF] Lindstrom | Alveolar breath sampling and analysis to assess trihalomethane exposures during competitive swimming training[END_REF] leading to several diseases as chronic lung diseases and nephrological diseases ( ; ). Erythrocytes Sies 1985 [START_REF] Halliwell | Protection against oxidants in biological systems: the superoxide theory of oxygen toxicity . Free Radicals in Biology and Medicine[END_REF] are circulating antioxidant carriers that can penetrate lung capillaries and have been shown to play a significant role in protection against lung damage due to ROS ( ; ;

). Toth et al. 1984Van Asbeck et al. 1984[START_REF] Agar | Erythrocyte catalase: a somatic oxidant defence[END_REF] Among the few studies which have been involved in the variations of the markers of response to oxidative stimuli in swimmers, none of them have studied the effect of exposure to chlorinated compounds ( ; ; ; [START_REF] Dickson | Effects of ultra-marathon training and racing on hematologic parameters and serum ferritin levels in well-trained athletes[END_REF]Lukaski 1989Lukaski et al. 1990 Inal et al. ; ). Moreover, none of them have measured more than two markers of response to oxidative stimuli, their 2001 Santos-Silva et al. 2001 control groups were usually non-exposed and non-training groups, and age ranges and sex were not the same. Finally, they have investigated different ways of training. It s well known that physical exercise was associated with response to oxidative stimuli in two '

ways (

). On one hand, exercise increased oxidative metabolism and this induced oxidative stress, but on the other hand, [START_REF] Moller | Oxidative stress associated with exercise, psychological stress and life-style factors[END_REF] the adaptation to regular exercise seemed to have an antioxidant protective effect.

In this study, we investigated the relations between chronic exposure to chlorinated compounds with several markers of response to oxidative stimuli in swimmers and we better understood these relations in taking into account the effect of training. Swimmers were surveyed twice: one week without exposure and one week with exposure to chlorinated compounds.

Material and methods

Study sample

Our study was performed in an indoor swimming pool located in Nancy (north east France). The participation was proposed to all male swimmers from the same club, training exclusively in this swimming pool during the season with the following criteria: the swimmers must be 15 25 years old and train regularly (at least once a day) without training the last week and the last weekend before examination. The participation rate was 80 . Our study sample was composed of 22 swimmers. Swimmers were examined twice the week % after the draining of the swimming pool (week0 (W0), day1 (D1) and day4 (D4)) and twice 24 weeks later, before the draining of the swimming pool (week24 (W24), D1 and D4). In April 1999, works of renovation began in the swimming pool of our study. A new technique of water treatment with new pumps and the installation of goulottes finlandaises were realized, leading to a decrease in the " " concentration of chlorinated compounds ( ). Among the 22 swimmers, 8 have benefited from these improvements (group [START_REF] Hery | Exposure to chloramines in the atmosphere of indoor swimming pools[END_REF] 2) and 14 have not (group 1). This study was approved by the appropriate ethical committee and a written informed consent obtained from each swimmer.

Examination and measurements of training and exposure

Each examination included a questionnaire on medical history, medication, dietary and smoking habits, alcohol consumption and sport activities. The frequency of consumption of fruits and of green or red vegetables (raw or cooked) was obtained from the questionnaire. The dietary score was the mean of the three scores obtained at each question. Training was defined as swimming and other aquatic and non aquatic sport activities. The number of years of swimming, the number of hours of swimming the last week and the last weekend before the week of examination were recorded. The number of hours of swimming between day1 and day4 and the number of hours of sports activities were also recorded.

Exposure to disinfection by-products was evaluated by the chloramine concentration in the atmosphere of the swimming pool and by the presence of trihalomethanes (THM) in the blood of the swimmers. Chloramine concentration was measured by the technique of Hery et . A feasibility study performed in this swimming pool during the 1997 1998 season showed a difference in the atmospheric al. (1995) chloramine concentration between week0 and week24 (0.17 mg/m3 vs. 0.49 mg/m3). The exposure assessment campaign was conducted systematically during day1 and day4 at each week of examination. Series of 6 to 10 samples were taken at the water surface in order to be representative of the swimmers exposure to chloramines. Special care was taken to avoid the filters to be splashed. The sampling duration was from 2 to 3 hours, representative of the exposure during the swimming session and providing a sufficient analytical sensitivity.

Trihalomethane determinations were conducted using the headspace technique with a gas chromatograph equipped with a split injector and an Ni electron capture detector ( ).

63 [START_REF] Ambroise | Evaluation du risque toxique li la pr sence de trialom thanes dans l eau utilis e pour la dialyse é à é é ' é[END_REF]

Biological markers of response to oxidative stimuli

At each week, blood samples were collected twice before swimming, at day1 and day4. On the same day, corresponding haemolysates, plasma and serum were prepared as described previously ( ), and stored at 80 C. Perrin et al. 1990 3 8

Total plasma antioxidant concentration (ascorbate, protein thiols, bilirubin, urate and tocopherol), Cu /Zn superoxide dismutase α ++ ++

(Cu /Zn SOD), glutathione peroxidase (GSH-Px) and catalase activities were measured by methods previously described (

++ ++
Perrin et al.

;

). Activities were measured in the two weeks following storage and expressed as U/g Hb (Cu /Zn SOD, 1990 [START_REF] Nadif | Relations between occupational exposure to coal mine dusts, erythrocyte catalase and Cu /Zn ++ ++ superoxide dismutase activities, and the severity of coal workers pneumoconiosis[END_REF] ++ ++ GSH-Px) or k/g Hb (catalase) in erythrocytes, and U/L (GSH-Px) in plasma.

Ferritin concentration was measured at 37 C at 600 nm in a Cobas-Mira S analyser with a Roche kit (ABX, Montpellier, France).

°Results were expressed as ng/ml. Ceruloplasmin concentration was measured at 37 C at 340 nm in a Cobas-Mira S analyser with a Roche °kit (ABX, Montpellier, France). Results were evaluated by comparing calibrators of known concentrations and were expressed as g/l.

All samples were analyzed in duplicate or triplicate and the precision (coefficient of variation) was <10 . The accuracy was checked % by analyzing external reference samples together with the test samples.

Statistical analyses

Standard Kappa coefficient for qualitative variables and intraclass correlation coefficient for quantitative variables. Swimmers were classified into subgroups according to their tertiles of the number of hours of swimming between day1 and day4 ( 4.2, from 4.2 to 9, >9).

≤

To describe the factors associated with changes in biological markers of response to oxidative stimuli taking into account the two follow-up factors, exposure and training, mixed models were used with the MIXED procedure. Confounding factors were put into the model. Validity of the models was checked with the null model likelihood ratio test and with the number of iterations of the criteria of convergence. Although the number of swimmers included in the multivariate analyses was small (n 16), it was sufficient to achieve a = power of 80 .

%

Significance was assessed at the 5 two-sided level. All these analyses were performed with SAS statistical software (

% SAS Institute, ). Cary, North Carolina, 2000

Results

Characteristics of swimmers and prevalence of irritation symptoms

Among the 22 swimmers who took part in the study, 16 have returned two questionnaires. The concordance for responses of past events between W0 and W24 was acceptable (all values were higher than 0.6, Kappa or intraclass correlation coefficient). Then, only the questionnaire returned at W0 was taken into account for past events for all swimmers.

Characteristics of the swimmers at W0 were described in . As expected, their mean age was between 15 and 25 years and their table 1 number of years of swimming was at least one year. Their alcohol consumption corresponded approximately to 0.25 liter of red wine per day. This consumption was 1.4 fold time lower than that of the French male consumption aged 18 24 years (

).

- [START_REF] Guilbert | Editor: Baudier F , Ar nes è J . Alcool: profils de consommation. Barom tre sant adultes 95/96 è é[END_REF] Irritant eye symptoms (red, burning or weary eyes), irritant nasal symptoms (running, burning nose or continued sneezing), throat irritations (sore throat or dry cough) and asthma were reported in . In absence of control population, these values were compared table 2 with those obtained in a group of 256 male lifeguards ( ) also exposed to chlorinated compounds. The prevalence of [START_REF] Massin | Respiratory symptoms and bronchial responsiveness in lifeguards exposed to nitrogen trichloride in indoor swimming pools[END_REF] irritant nasal symptoms, throat irritation and asthma were significantly higher in swimmers.

Swimmers surveyed twice

Based on available data, comparisons of swimmers lost to follow-up (n 6) and the remainder (n 16) did not show difference regarding dietary score was 2.5 (0.9) and their alcohol consumption was 13.3 g/d (14.1). Sixty seven percent never drank aperitif, 13 drank % between 1 and 4 aperitifs a month and 20 drank more than 4 aperitifs a month. Comparison of sport activities between W0 and W24 in % swimmers surveyed twice showed that the number of hours of swimming between D1 and D4 and the number of hours of swimming the last week were significantly higher in W24 than in W0.

Comparison of markers of response to oxidative stimuli between D1 and D4 showed several significant differences between both weeks ( ). At W0, erythrocyte Cu /Zn SOD activity and GSH-Px activities were significantly increased in D4. Moreover, table 3 swimming the last week (r 0.66, p 0.007; r 0.67, p 0.007, respectively). At W24, erythrocyte Cu /Zn SOD activity was also

=- = =- = ++ ++
significantly increased in D4 but no significant increase in erythrocyte GSH-Px activity was observed. At D4, Cu /Zn SOD activity

++ ++
was significantly and positively related to the number of hours of non-aquatic sport activities (r 0.98, p 0.004). On the contrary, plasma

= =
GSH-Px activity and ceruloplasmin concentration were significantly lower in D4.

Among the 11 swimmers surveyed twice at W24, 80 of those with a number of hours of swimming between D1 and D4>9 had marks % of THM in plasma (data not shown). Highly-exposed swimmers (marks of THM in plasma and hours >9) were younger, non-smokers, all of them were current drug users and their current drug consumption was significantly higher than lowly-exposed swimmers (no mark of THM in plasma and hours 9). Highly-exposed swimmers were all from the group 1. Most of them swam each day between blood ≤ collections and their number of hours of swimming the last week were higher than those of the low-exposed swimmers. As expected, highly-exposed swimmers had a number of hours of swimming between D1 and D4 two fold time significantly higher than lowly-exposed swimmers (11.6 vs. 5.4 hours, p 0.06) and plasma THM concentrations were higher in highly-exposed swimmers in D1 and in D4 (1.3

= ±
2.6 g/l vs. 0.7 1.4 g/l; 0.6 0.4 g/l vs. 0.4 1.1 g/l, in D1 and D4 respectively). No significant difference was observed in biological

μ ± μ ± μ ± μ
markers of response to oxidative stimuli between both groups of exposure at D1. Erythrocyte catalase activity was significantly increased in D4 in highly-exposed swimmers (147 27 k/g Hb vs. 175 37 k/g Hb, in D1 and D4 respectively, p 0.03) and plasma GSH-Px activity

± ± =
was decreased in D4 in both groups with a significant difference in lowly-exposed swimmers (662 69 U/l vs. 623 58 U/l, in

± ±
lowly-exposed swimmers in D1 and D4 respectively, p 0.05).

=

Multivariate analysis

Multivariate analyses were performed to study the associations between biological markers of response to oxidative stimuli and exposure, including the effect of training ( ). table 4 Only erythrocyte Cu /Zn SOD and GSH-Px activities were found to be significantly associated with exposure and training.

++ ++

Several interactions were found. The difference in the level of erythrocyte GSH-Px activity between the group 1 and the group 2 depended on the week of examination and on the number of hours of swimming between D1 and D4. In group 2, erythrocyte GSH-Px activity decreased with the number of hours of swimming between D1 and D4 and was higher in W24. The difference in the level of plasma GSH-Px activity in W24 depended on day of measurement and on age. In W24, plasma GSH-Px activity was lower in oldest swimmers and was lower in D4 than in D1, which may partly explain the results observed at W24 in all, as in highly-and lowly-exposed swimmers.

Discussion

To our knowledge, this study is the first to describe associations between biological markers of response to oxidative stimuli not only with training but also with exposure to chlorinated compounds in swimmers. In this study, we found that erythrocyte Cu /Zn SOD ++ ++ activity was related to training and exposure to chlorinated compounds and that GSH-Px activities were related to exposure to chlorinated compounds.

We found no change in catalase activity and ceruloplasmin concentration with exposure nor with training. Our results are consistent with those of other studies ( ; ; ; ; ). We found a [START_REF] Buchman | The effect of a marathon run on plasma and urine mineral and metal concentrations[END_REF][START_REF] Ji | Antioxidant enzyme response to exercise and aging[END_REF][START_REF] Marzatico | Blood free radical antioxidant enzymes and lipid peroxides following long-distance and lactacidemic performances in highly trained aerobic and sprint athletes[END_REF][START_REF] Inal | International Agency for Research on Cancer (IARC) . Chlorinated drinking water; chlorination by-products; some other halogenated compounds, cobalt and cobalt compounds[END_REF][START_REF] Miyazaki | Strenuous endurance training in humans reduces oxidative stress following exhausting exercise[END_REF] weak increase in ferritin concentration after training, as previously described by and . We also [START_REF] Lukaski | Physical training and copper, iron, and zinc status of swimmers[END_REF][START_REF] Dickson | Effects of ultra-marathon training and racing on hematologic parameters and serum ferritin levels in well-trained athletes[END_REF] reported a weak decrease in total plasma antioxidant concentration with exposure. In contrast, reported an increase Santos-Silva et al. 2001 in total plasma antioxidant concentration in swimmers. Our results were not confirmed by multivariate analysis and difference in design with study previously mentioned did not allow to discuss the results.

The increase in Cu /Zn SOD activity we found in D4 at each week is in agreement with other studies. have ++ ++ [START_REF] Miyazaki | Strenuous endurance training in humans reduces oxidative stress following exhausting exercise[END_REF] reported an increase in Cu /Zn SOD activity in 9 healthy, untrained and 19 21 years old male subjects after a 12 week running ++ ++ -

program.

have reported that basal Cu /Zn SOD activity was higher in athletes (6 marathon runners aged 26. 8 Marzatico et al. (1997) ++ ++ years and 6 national sprint-trained athletes aged 25.2 years) as compared to 6 healthy males aged 28.5 years and normally physically active. They also found that Cu /Zn SOD activity was significantly increased in the runners and in the sprinters after a training session

++ ++
and suggested that this result could reflect a training effect. Finally, they concluded that a potential oxidative stress was generated by daily training sessions. Only one follow-up study has investigated the effect of training on markers of response to oxidative stimuli in swimmers ( ). The authors found an increase in erythrocyte Cu /Zn SOD activity in 13 male swimmers after 6 months of [START_REF] Lukaski | Physical training and copper, iron, and zinc status of swimmers[END_REF]

++ ++

training. They also found that before and after the training season, Cu /Zn SOD activity was higher in swimmers than in age-matched

++ ++
untrained controls and concluded to a training effect. We found that Cu /Zn SOD activity was also increased by exposure. In spite of

++ ++
no information on the exposure to chlorinated compounds in the swimming pool of the study of (draining, chlorinate Lulaski et al. (1990) rate, etc.), we may not exclude that the training effect they observed was in fact a combined effect of training and exposure. Moreover, " " they didn t perform any multivariate analyses taking separately into account the effect of exposure and the effect of training. In our study,

'
the relation between Cu /Zn SOD activity and exposure was reinforced by the fact that Cu /Zn SOD activity was higher in group

++ ++ ++ ++
1 than in group 2. One may not explain the difference between group 1 and group 2 by a difference in one or several characteristics of the swimmers or in their ways of training because these two groups were totally similar regarding to these covariables and the only difference we know was the water treatment. The difference we observed between the groups could be partly due to a difference in exposure to chlorinated compounds. All these results well supported the hypothesis that exposure and training were both involved in the increase of the Cu /Zn SOD activity.

++ ++

We found several variations in GSH-Px activities, mostly with exposure. previously reported that erythrocyte [START_REF] Inal | International Agency for Research on Cancer (IARC) . Chlorinated drinking water; chlorination by-products; some other halogenated compounds, cobalt and cobalt compounds[END_REF] GSH-Px activity was higher one minute after both long-distance and short-distance swimming in 19 male or female aged 15 21 years than at rest. and also observed an increase in erythrocyte GSH-Px activity after training with a concomitant [START_REF] Marzatico | Blood free radical antioxidant enzymes and lipid peroxides following long-distance and lactacidemic performances in highly trained aerobic and sprint athletes[END_REF][START_REF] Ji | Antioxidant enzyme response to exercise and aging[END_REF] increase in erythrocyte Cu /Zn SOD activity. We showed that Cu /Zn SOD activity was related to training. Our explanation is

++ ++ ++ ++
that the increase in erythrocyte GSH-Px activity we observed in D4, as those observed in the studies of and of [START_REF] Marzatico | Blood free radical antioxidant enzymes and lipid peroxides following long-distance and lactacidemic performances in highly trained aerobic and sprint athletes[END_REF] Ji

was in fact a response to an increase in erythrocyte Cu /Zn SOD activity. The increase in Cu /Zn SOD activity leads to a (1993)

++ ++ ++ ++
production of H O following by an increase in GSH-Px activity ( ). This is underlined by the positive 2 2

Halliwell and Gutteridge 1989 correlation we found between erythrocyte Cu /Zn SOD and GSH-Px activities in W0 (r 0.39, p 0.01). In W24, we found a decrease

++ ++ = =
in erythrocyte GSH-Px activity with exposure and a negative correlation between erythrocyte Cu /Zn SOD and GSH-Px activities (r between plasma and cytosolic GSH-Px and the fact that both biological compartments are distinct, may explain these results [START_REF] Harris | Regulation of antioxidant enzymes[END_REF] ). Nevertheless, we cannot exclude that the positive association between plasma GSH-Px and exposure found in the multivariate analysis could reflect in part a compensatory mechanism of the lack of increase in erythrocyte GSH-Px activity. As we are the only study dealing with the relation between exposure and plasma GSH-Px, this is open to debate.

Another interesting finding that indirectly supported the hypothesis of a production of reactive species from exposure to chlorinated compounds was the higher prevalence of eye, nasal and throat irritant symptoms observed in swimmers than in lifeguards. Among lifeguards, the prevalence tended to increase with increasing exposure to nitrogen trichloride (NCl ) ( ). The lifeguards 3 [START_REF] Massin | Respiratory symptoms and bronchial responsiveness in lifeguards exposed to nitrogen trichloride in indoor swimming pools[END_REF] worked essentially outside the water, in the surrounding area of the pool and the swimmers were directly exposed to chlorinated compounds on the pool water surface. Exposure to higher concentration of NCl could explain higher prevalence reported in swimmers.

3 studied 14 competitive swimmers and 14 matched control subjects. Conjonctival or respiratory symptoms (rhinitis, Zwick et al. (1990) laryngitis and bronchitis) were founded in 11 swimmers and in 3 controls. They concluded that this high prevalence could be due to repeated exposure to chlorine in swimming pools. We also found a high prevalence of asthma in swimmers. studied [START_REF] Nystad | Asthma and wheezing among Norwegian elite athletes[END_REF] asthma among elite athletes included all Norwegian athletes of the national junior and senior teams in 1997 (n 1620) and a random sample = from the general population (n 1680). Their results indicated that the prevalence of asthma was greater among athletes (10 ) as compared = %

to the general population (6.9 ) and that the risk of asthma was the highest in sport requiring strength and endurance.

%

Helenius and supported the same judgment after reporting that asthma is most commonly found in endurance events, such as cycling, Haahtela (2000) swimming, or long-distance running. But they also found that the risk of asthma was especially increased among competitive swimmers of which 36 to 79 show bronchial responsiveness to methacholine or histamine. High or intensive training but also exposure to % % chlorinated compounds could explain this high prevalence of asthma.

In conclusion, results reported in this study are in good agreement with the hypothesis that the production of reactive oxygen species is not only related to training but also related to exposure to chlorinated compounds, and that ROS may partly explain the toxicity of chlorinated compounds.

Table 3

Comparisons of biological markers of oxidative stress between D1 and D4 at each week in swimmers surveyed twice

  and exposure. Swimmers in whom data were available at both weeks (n 16) were 19.8 (3.8) years old, = non-smokers and had a mean number of swimming of 4.4 years. Half of them currently took drugs and all of them took vitamins. Their

  The response to ROS exposure was biologically measured with specific and sensitive blood markers (catalase, Cu /Zn superoxide dismutase (Cu /Zn SOD), were examined twice before swimming in order to dismiss the acute effects of exposure or/and training. Training was assessed as swimming and other sport activities.

	++	++	++	++
	glutathione peroxidase (GSH-Px), total plasma antioxidants, serum ferritin and ceruloplasmin) (	Halliwell and Gutteridge 1989	). At each
	week, swimmers

  statistical tests were used including X test (or Fisher s exact test when appropriate), McNemar s test, Student s test and

	2	'	'	' t
	Pearson s correlation test ( ). Concordance between questionnaires for variables concerning swimmer s history was quantified using

' r '

  /Zn SOD activity to be overwhelmed by the high production of O , and the excess of O able to inactivate GSH-Px activity ( Zn SOD and plasma GSH-Px activities was observed in W0 nor in W24. A difference in the homology

							++	++	=-
	0.41, p 0.03) and we found that erythrocyte Cu /Zn SOD activity was positively related to exposure and training. The capacity of Cu = ++ ++	+
	+	++		2	. -	2	. -	Blum
	and Fridovich 1985	) could explain our results. A similar inactivation of GSH-Px activity was not observed in plasma and no correlation
	between erythrocyte Cu /++	++

  Results are expressed as mean (SD) except for ferritin (geometric mean).

	3
	In group 1/2	0.27/0.25	0.27/0.24	-	-	0.32/0.30	0.19/0.30	-	-
	In erythrocytes:
	Catalase (k/g Hb)	160 (37)	173 (31)	3.11 (42.50)	0.8	157 (27)	161 (28)	3.60 (27.18)	0.7
	Cu /Zn SOD (U/g Hb) ++ ++	152 (18)	176 (19)	21.6 (17.5)	0.0001	173 (22)	187 (25)	21.7 (23.6)	0.01
	GSH-Px (U/g Hb)	40.9 (9.4)	42.7 (10.3)	2.36 (3.68)	0.02	33.8 (7.3)	34.0 (6.7)	0.38 (2.75)	0.7
	In plasma:
	GSH-Px (U/L)	567 (119)	631 (101)	61.5 (77.1)	0.005	653 (83)	635 (57)	-46.5 (48.5)	0.01
	Total antioxidant concentration (mmol/L)	1.40 (0.07)	1.38 (0.07)	-0.007 (0.061)	0.6	1.37 (0.11)	1.34 (0.10)	-0.01 (0.10)	0.6
	In serum:
	Ferritin (ng/mL)	55.3	56.2	0.11 (0.22)	0.06	36.3	46.5	0.11 (0.45)	0.5
	Ceruloplasmin (g/L)	0.19 (0.02)	0.19 (0.03)	0.003 (0.017)	0.6	0.19 (0.02)	0.18 (0.02)	-0.007 (0.008)	0.03

Table 4

 4 Factors associated with biological markers of oxidative stress in swimmers surveyed twice

Erythrocyte Cu /Zn SOD (U/g Hb)

		++	++	Erythrocyte GSH-Px (U/g Hb) Plasma GSH-Px (U/L)	Total antioxidant concentration (mmol/L)
		Coefficient (SD)	p Value	Coefficient (SD)	p Value Coefficient (SD) p Value Coefficient (SD)	p Value
	Exposure: W24 vs. W0	16.1 (5.28)	0.01	-15.3 (1.37)	0.002	246 (52.7)	0.002	-0.038 (0.021)	0.09
	Training: D4 vs. D1	19.3 (3.49)	0.0001	-0.11 (1.17)	0.9	71.0 (27.3)	0.3	-0.014 (0.021)	0.5
	Group: 2 vs. 1	-25.8 (4.53)	< 0.0001	-8.29 (4.19)	0.9	-4.44 (25.7)	0.9	-0.003 (0.032)	0.9
	Hours of swimming between D1 and D4			0.24 (0.33)	0.15
	Hours of swimming between D1 and D4 group 2 *			-1.47 (0.58)	0.06
	Exposure group 2 *			15.5 (2.38)	0.0009
	Age					14.2 (4.42)	0.01
	Exposure age *					-6.99 (2.69)	0.02
	Exposure D4 *					-94.0 (16.2)	< 0.0001

Aiking H , Van Acker MB , Scholten RJPM , Feenstra JF , Valkenburg HA . Swimming pool chlorination: a health hazard? . ToxicolLett . 1994 ; 72 : 375 -380

Ackowledgements:

The paper is dedicated to the late Jean-Marie Gerber. We thank all the swimmers and Alain Chaxel, Anne Cochart, Jean-Claude Glaudel, Jacky Macadre, Fr d ric Muel, Claude Pourchet and Marie Roure who made this study possible, and Bruno Falissard for helpful comments.

é é

This research was, in part, supported by grant number AC020C from the Minist re de l Am nagement du Territoire et de l Environnement.

> 4 drinks/month 13 (65) * means of the three scores of dietary habits ranging from 1 (never) to 4 (at least once a day). % * data obtained from using same questions. [START_REF] Massin | Respiratory symptoms and bronchial responsiveness in lifeguards exposed to nitrogen trichloride in indoor swimming pools[END_REF]


Philippe Lefebvre

Yacir Benomar

Bart Staels

Retinoid X Receptors: common heterodimerization partners with distinct functions

Nuclear receptor biology and pathophysiology studies have emphasized the roles of retinoid X receptors (RXRs) in cellular processes ranging from cellular proliferation to lipid metabolism. These pleiotropic effects stem not only from RXR=s ability to dimerize with various nuclear receptors, which exert transcriptional control on specific aspects of cellular biology, but also from the permissive nature of some RXR heterodimers, reflecting the ability of RXR ligands to activate this heterodimer. This signaling network is rendered more complex by RXR isotypes (RXRα, RXRβ, RXRγ) which have some distinctive properties, thereby modulating RXR-containing heterodimer transcriptional activities. This review discusses the emerging roles of RXR isotypes in the RXR signaling network, with possible implications in our understanding of nuclear receptor biology and pharmacology.

Overview of RXR features

The retinoid X receptors (RXRs) are encoded by three distinct genes located on human chromosomes 9 (RXRΑ, NR2B1), 6 (RXRB, NR2B2) and 1 (RXRG, NR2B3), or on mouse chromosomes 2, 17 and 1 respectively, and share strong homologies in their coding sequences.

RXRs have been initially characterized as heterodimeric partners of the retinoic acid (RARs), thyroid hormone (T3Rs) and vitamin D (VDR) nuclear receptors (NRs) and are required for high affinity binding to DNA of these and other RXR heterodimers 1 . NRs heterodimerizing with RXRs define a subclass which can be activated, or not, through RXR agonism. This (non)permissivity of RXR heterodimers underlines the central and complex roles of RXR in multiple biological processes and also the difficulty for precisely assessing their importance. The realization that RXRs could form homotetramers in solution and bind to DNA as homotetramers and homodimers [START_REF] Yasmin | Inhibition of mammary carcinoma cell growth by RXR is mediated by the receptor's oligomeric switch[END_REF][START_REF] Ijpenberg | In vivo activation of PPAR target genes by RXR homodimers[END_REF] led to the speculation that RXRs may control their own specific signaling pathways, rendering the analysis of the RXR pathway even more complex. Initial genetic analysis of RXR functions essentially pointed to their roles in embryonic development, which is believed to be conveyed mostly through RXR-RARs , and driven by the activation of RARs by the morphogen molecule all trans retinoic acid (atRA)

. Whereas the biological relevance and functions of atRA are nowadays undisputed, the 9-cis retinoic acid isomer, which was initially identified as a bona fide RXR ligand in vitro 1 , has never been

3

detected in vivo 6

. This prompted the search for novel natural ligands, among which the unsaturated fatty acid docosahexanoic acid (DHA) [START_REF] Deurquiza | Docosahexaenoic acid, a ligand for the retinoid X receptor in mouse brain[END_REF] and -apo14'-carotenal [START_REF] Ziouzenkova | Asymmetric cleavage of beta-carotene yields a transcriptional repressor of retinoid X receptor and peroxisome proliferator-activated receptor responses[END_REF] activate or repress RXR transcriptional activity respectively. However, these natural molecules are not RXR-specific ligands (rexinoids) and regulate notably the transcriptional activity of peroxisome proliferator-activated receptors (PPARs). The synthesis of rexinoids has revealed their potential as chemotherapeutic agents, exemplified by the successful use of Targretin7 (bexarotene) in T-cell lymphoma treatment 9 .

However, the promiscuous nature of RXRs has hindered the further development of rexinoids, especially in light of their propensity to cause hypertriglyceridemia 10 , a risk factor of cardiovascular diseases in spite of their beneficial effects on insulin sensitivity and fat mass.

As for all other NRs, the RXR ligand binding domain folds into a three-layered -helical sandwich defining a hydrophobic ligand-binding pocket (LBP). In the unliganded state, the AF-2 AD (helix 12) is exposed to the solvent and yields free access of the ligand to the LBP (Figure 1). The RXR LBP is defined by the amino termini of helices 3 and 9, the two beta sheets, helix 8, the H8-H9 loop, and the carboxyl terminus of helix 11

11

. In analogy with RARs, the synthesis of RXR isotype-specific ligands would have been highly desirable. Unfortunately, the strong conservation of LBD and of LBP (ca. 500

3

) between RXR isotypes does not allow an easy design of isotype-selective ligands. However, the design of heterodimer-selective rexinoids [START_REF] Mukherjee | Sensitization of diabetic and obese mice to insulin by retinoid X receptor agonists[END_REF][START_REF] Cesario | The rexinoid LG100754 is a novel RXR:PPARgamma agonist and decreases glucose levels in vivo[END_REF][START_REF] Morita | Selective allosteric ligand activation of the retinoid X receptor heterodimers of NGFI-B and Nurr1[END_REF] demonstrated that activation of permissive heterodimers through RXR is a viable option for the pharmacological and possibly therapeutical control of dysregulated cellular pathways

15

. Furthermore, the control of RXR heterodimer non-permissivity through the activation of kinase signaling pathways (desubordination)

provides another tool to modulate RXR heterodimers transcriptional activity 16 . In this review, we will discuss RXR biology and highlight recent advances in this field to forecast how this increased knowledge may create new options for drug discovery.

RXR isotypes: similar but different.

Tissue-specific distribution. The Rxra, Rxrb and Rxrg genes are differently expressed in mouse tissues: Rxra is the major isotype in mouse liver, whereas Rxrb expression is highest in the central nervous system. Rxrg displays a more restricted expression territory, with detectable levels in skeletal muscle and regions of the CNS such as the olfactory bulb and the pituitary glands

4

RXRs are expressed as 3 isotypes and numerous isoforms resulting mostly from alternative splicing (Figure 1). The predominant mouse RXRα isoform is RXRα1, which encodes a 52kDa receptor. Two additional isoforms have been isolated from adult mouse testis, RXRα2 and RXRα3, which have a 28 and a 97 amino acid deletion in the N-terminal AF1 domain respectively [START_REF] Brocard | Two novel RXR alpha isoforms from mouse testis[END_REF]

RXR, a versatile dimerization partner.

RXR dimers: RXRs establish dimeric complexes either spontaneously [START_REF] Feige | Fluorescence imaging reveals the nuclear behavior of peroxisome proliferator-activated receptor/retinoid X receptor heterodimers in the absence and presence of ligand[END_REF] or in a ligand-dependent manner [START_REF] Depoix | Control of retinoic acid receptor heterodimerization by ligand-induced structural transitions. a novel mechanism of action for retinoid antagonists[END_REF] with a number of other NRs (Figure 2 . Of note, a number of proteins involved in various biological processes have been shown to interact physically and/or functionnally with RXRs (Figure 2), paving the way for the discovery of novel RXR functions. The interaction of RXR with NPAS/MOP4 and Clock, two critical regulators of the circadian feedback loop 67 , suggest for example that circadian rhythms can be brought under the control of endogenous RXR ligands such as DHA, whose availability may depend on the nutritional status.

As previously documented, NR-mediated transcription involves DNA double strand breakage and DNA repair [START_REF] Kress | Active cytosine demethylation triggered by a nuclear receptor involves DNA strand breaks[END_REF][START_REF] Ju | A topoisomerase IIbeta-mediated dsDNA break required for regulated transcription[END_REF] and in this context, another role of RXR that could also be envisioned is the control of DNA repair through physical interaction with the T:G mismatch-specific thymine-DNA glycosylase (TDG, 70

), in analogy with the reported PCNA-RARα interaction 71 .

RXRs= ability to homotetramerize or homodimerize has been proposed to be, at least in part, the molecular basis for anticarcinogenic or metabolic properties of rexinoids respectively . These data show that RXR isotypes are involved in a plethora of biological responses which are tissue-and very likely isoform-specific. A more thorough investigation using conditional, tissue-specific KO mice would undoubtedly bring new insights into this complex RXR biology.

RXR permissivity and metabolic regulations

Although highly dependent on the model system used to assess this property, RXR-containing heterodimers can be categorized, quite schematically, as either Apermissive@ or Anon-permissive@, as they can be activated, or not, through the RXR moiety. Nuclear receptors with a high affinity for their cognate ligands are mostly found in the non-permissive category, such as RARs, T3R and VDR

9

heterodimer through RXR does not equate activation through the RXR partner: the transcriptional response elicited through RXR overlaps only partially with that elicited through its partner 86-88 . In a detailed study, it was found that activation of LXR target genes in liver by a rexinoid was restricted to genes involved in lipogenesis, whereas those involved in cholesterol homeostasis were left unaffected.

While providing a molecular explanation for the hypertriglyceridemic effect of bexarotene and possibly other rexinoids, these data raise the yet unresolved question of whether this reflects a selective permissivity of RXR-LXR heterodimer or another mechanism 89 . This may relate to the differential affinity of LXR and RXR for LXXLL-containing peptides [START_REF] Son | RXR heterodimerization allosterically activates LXR binding to the second NR box of activating signal co-integrator-2[END_REF] and thus to the assembly of distinct coactivator complexes on each receptor monomer. Alternatively, selective post-translational modifications may control RXR permissivity in a promoter-specific context, in analogy to the protein kinase A-dependent desubordination of RXR in the RXR-RAR heterodimer 16 .

Towards heterodimer-specific rexinoids

RXRs can bind a variety of natural and synthetic derivatives, ranging from ω-3 unsaturated fatty acids, the non steroidal anti-inflammatory drug sulindac to organotins

10

after a seven day treatment [START_REF] Michellys | Novel (2E,4E,6Z)-7-(2-alkoxy-3,5-dialkylbenzene)-3-methylocta-2,4,6-trienoic acid retinoid X receptor modulators are active in models of type 2 diabetes[END_REF] . However, RXR antagonism in the RXR-PPARγ dimer has also proven to be efficient in normalizing glycemia and fat mass in animal models 97 , but this pharmacological option has not been explored further. Taken together, these data suggest that the design of heterodimer-specific rexinoids can be envisioned, although no precise rules have emerged yet for an efficient drug design.

The RXR cistrome

The recent availability of deep sequencing techniques led to the identification of RXR dimer DNA binding sites on a genome-wide scale. A consistent finding was that these potential regulatory sites are very often located at intronic locations at a distance from the transcriptional start site of regulated genes, calling for the re-examination of previous promoter studies which identified functional response elements in the vicinity of promoter regions. Another observation was that a vast majority of these heterodimer binding sites are prebound by RXR and that either ligand treatment or increased expression of the RXR partner lead to the expected heterodimer formation. This suggests a model in which RXR serves as a docking site for multiple nuclear receptors, and that the heterodimer composition will vary according to extra-or intracellular cues activating or repressing RXR and/or its dimerization partner. Furthermore, these data provide a general ground for the widely held assumption that RXR increases the DNA binding affinity of RXR-containing heterodimers 98-100

.

Concluding remarks

Heterodimerization is a general, simple mechanism by which a linear signaling pathway can be converted into a very complex network acting in a cell-specific manner in response to physiological or pathological cues. RXRs act as common heterodimerization partners for a number of other nuclear receptors, and as such are often considered as loosening the specificity of the signaling pathway. As a consequence, their usefulness as drug targets has been dismissed. However, the occurrence of several isotypes, and of multiple isoforms raise the possibility, as demonstrated by some reports described above, that RXR functions may be diverse and mediated through distinct molecular entities.

An in-depth understanding of their respective biological functions, as well as the identification of

11

heterodimer-selective synthetic compounds, will undoubtedly unravel novel aspect of RXR biology and pharmacology.

Future directions box:

$ Is there specific, endogenous ligands for RXR?

$ Can these ligands regulate specific metabolic pathways?

$ What are the functions for each RXR isotype?

$ Are RXRs only transcriptional regulators?

4

 4

62 . 63 .

 6263 ), resulting in distinct functional outcomes. While necessary for high affinity DNA binding of several NRs such as the T3R, vitamin D or RARs, FXR/RXR dimers display a strongly decreased DNA binding upon rexinoid ligation 60 . The DNA binding motifs to which RXR heterodimers bind are, in most cases, direct repeats (DR) containing the AGGTCA heptameric sequence and follow the so called 1 to 5 rule. According to this rule, RXR homodimers and PPAR-RXR heterodimers bind to DR1 motifs, whereas DR2 and DR5 are preferential binding motifs for RXR-RAR heterodimers, and DR3 and DR4 favor the association of vitamin D3 and T3 receptor heterodimers, respectively. This rule is however quite flexible since, for example, FXR-RXR dimers can also bind to inverted repeat (IR) motifs 61 and RXR-RAR dimers to DR4 motifs It is however clear that the polarization of RXR heterodimers plays a crucial role in the transcriptional outcome upon ligand activation RXR dimerization can also confer new biological properties: as mentioned above, RXR association with Nur77/NR4A1 promotes mitochondrial targeting of Nur77 and subsequent apoptosis 64 , whereas RXR association to the PML-RARα fusion protein unleashes PML-RARα oncogenic 7 potential in hematopoietic cells 65,66

79 . 80 . 83 . 84 .

 79808384 These receptors have a noticeable intrinsic repressive activity in the unliganded state. In contrast, low affinity-binding, lipid-activated nuclear receptors such as peroxisome proliferator-activated receptors (PPARs), the bile acid/farnesoid receptor (FXR) or oxysterol receptors (LXRs) are considered as permissive entities, suggesting that the functional control of RXR partners evolved to couple differentially endocrine and metabolic regulations The mechanism by which this molecular crosstalk takes place is still poorly defined, and is very certainly specific for each heterodimer: indeed, while the C-terminal activating function 2 (AF2, required for coactivator recruitment) of RXR is dispensable for PPARγ synergistic activation, it is required for PPAR and T3R activation81,82 . The exact physical basis for this functional synergy is unknown, however, mutagenesis studies and structural studies suggest that one coactivator molecule is recruited per heterodimer, with a single LXXLL motif being anchored to each partner Intriguingly enough, the structural studies dismissed the possibility of allosteric phenomenons between heterodimeric partners, at least for RAR-RXR heterodimers In contrast, mutagenesis studies of the RXR-LXR complex spoke in favor of allosteric interactions between the two components of the dimer 85 , a difference which could explain the permissive nature of the LXR-RXR dimer. Importantly, the activation of a given permissive

Figure 1 :

 1 Figure 1: RXR sequences and structures. A) RXR primary structure. RXR isotypes have a structural organization similar to that of other nuclear receptors but lack a C-terminal F domain. AF-1: Activation Function-1; DBD: DNA Binding Domain; LBD: Ligand Binding Domain; AF-2: Activating Function 2; AF2-AD: AF2 activation domain (corresponding to helix 12 of the LBD). Splice variant names are indicated on the left, while the structure of the encoded protein is indicated on the right. B) Tertiary structure of the unliganded (Apo-RXRα) and of the liganded RXRα LBD to docosahexanoic acid (Holo-RXRα) complexed to a SRC2 peptide. Coordinates were taken from the RCSB protein databank (1LBD and 1MV9) and visualized using the JMol software. C) Quaternary structure of a full length PPARγ-RXRα heterodimer. Coordinates (3DZY) and molecule structure were obtained as described in B).

Figure 2 :

 2 Figure 2: The RXRα interaction network. Proteins engaging physical and functional interactions with RXRα are indicated and classified according to their main functions. Data were compiled from the visANT database [Hu, Z. et al., 2007, Towards zoomable multidimensional maps of the cell. Nat Biotechnol, 25(5): 547-54] and a PubMed search.

  Rxra, Rxrb and Rxrg expression does not follow a rhythmic cycle of expression in metabolically active tissues from C57Bl/6 mice, with the exception of Rxra in liver, and in opposition to a number of other RXR dimerization partners such as residues is dependant on its association with RAR and can affect the transcriptional cooperation between RAR and RXR {Gianni, Tarrade, et al. 2003 18581 /id}. This non exhaustive list of the role of PTMs on RXR activity suggest a great flexibility of RXR functions according to pathophysiological conditions which affect regulatory signaling pathways.

	conditions the RXR-mediated targeting of Nur77/NR4A1 and of the thyroid hormone receptor to
	mitochondria to promote apoptosis	53,54 . Thus, in addition to a simple, on-off regulatory mechanism of
	RXR expression, more subtle mechanisms affecting its cellular localization control RXR transcriptional
	. Ensembl . Finally, Rxrg1 expression is down-regulated in pituitary cells by 9-cis-RA 35 activities and may generate novel functions for this multi-faceted nuclear receptor. Finally, fine-tuning FXR, PPARs and RARs
	database inspection also identifies a fourth splice variant 4 potentially generating a 165 aminoacid through an atypical DR1 response element 36 . of RXR transcriptional activity also relies on post-translational modifications (PTM): the
	protein which has not been characterized functionally. Similarly, four mouse RXRβ isoforms have In contrast, a number of reports documented the proteasomal degradation of RXRα in various MAPK-dependent phosphorylation of human RXRα at serine 260 regulates coactivator recruitment to
	been identified. The human homolog of mRXR1, hRXR2, is conserved across species but cellular backgrounds 37 , which appeared to be conditioned by RXR interaction with RAR and the a RXR-VDR dimer 55 and its degradation through the ubiquitin-proteasome pathway 56 , a PTM which
	contains two ATG initiator codons which may produce N-terminal variants corepressor SMRT 38,39 and increased upon agonist binding 40,41 . Moreover, RXRα breakdown is 19 . A third isoform, initially can be controlled through the growth factor status of target cells 57 . In addition, RXR phosphorylation
	termed hRXRβ3, contains a SLSR sequence inserted in the ligand-binding domain through the use of dependent on MAPK or PKC activity 42,43 , hinting at a link between RXRα phosphorylation and on distinct serine
	an alternative 3'-splice acceptor site, similar to the mouse RXRβ2E isoform proteasomal-mediated degradation	20 . This insertion impacts
	negatively on the ligand binding activity of RXRβ and on its transactivation potential. Finally, mRXRγ1
	is strongly expressed in the brain and muscle, whereas the mRXRγ2 isoform has a unique
	5'-untranslated region and is expressed highly in both cardiac and skeletal muscles	21 .
		The respective role(s) of RXR isotypes and isoforms is not fully understood. Germ line
	mutations in the Rxr genes induce either in utero lethality through congenital heart defects (RXRα), or
	metabolic and behavioral defects [RXRβ, RXRγ, reviewed in	22 ]. From a developmental point of view,
	RXRα seems to be the main player since mice expressing only RXRα from one allele are viable,
	indicating a strong functional redundancy	23 . Tissue-specific inactivation of Rxra in hepatocytes	24-28
	or epithelia	29,30 induces strong phenotypes, indicating a major role of RXRα in these tissues, whereas
	Rxra gene knockout has only a mild effect in hematopoietic progenitors
	nucleus	49,50 . The nucleoplasmic sublocalization to the splicing factor compartment may also be
	another factor controlling RXR transcriptional activities	51 . LPS treatment of target cells can induce
	both downregulation of Rxr expression and its cytoplasmic relocalization	52 , providing a molecular
	basis for the observed repression of RXR target genes during TLR4-mediated inflammation. This
	cytoplasmic relocalization likely depends on RXRα nuclear export signal located in its DBD, which also
				6

31

. The conditional ablation of RXRα in adipocytes revealed that this isotype is necessary for adipocyte lipogenesis and hypertrophy, a phenomenon observed in obese animals, but not for adipocyte survival, for which RXRγ can compensate for RXRα ablation 32 .

Regulation of RXR expression and cellular localization.

In view of their physiological importance, the mode of regulation of RXR gene expression has quite paradoxically received little attention. Only the cloning and characterization of the promoter of the human RXRA gene has been reported, revealing a TATA-less sequence with features of housekeeping genes 33 . Direct transcriptional regulation of RXRα,  and  gene expression is seen in severe sepsis and upon LPS 5 or cytokine treatment in rodent heart and liver 34 . Interestingly, 44 . The sensibility of RXRα to proteasomal degradation is selective, since RXRβ is resistant to proteasome-mediated degradation in human embryonic kidney (HEK) cells 42 , in mouse 3T3-L1 preadipocytes and in visceral mouse and human adipose tissues 45 . RXRα, but not RXRβ, is specifically conjugated to ubiquitin both in vitro and in vivo, leading to a marked, specific RXRα breakdown in obese visceral fat. Indeed, mouse and human obese visceral fat overexpress the ubiquitin hydrolase/ligase UCH-L1, which catalyzes, by a yet undefined molecular mechanism, RXRα ubiquitinylation and breakdown 45 . Quite similarly, RXRα is downregulated in many cancer cells and tissues and proteolytically degraded by cathepsin L and/or calpain, generating a cytoplasmic, N-terminally 44 kDa truncated receptor able to interact with the protein kinase Akt and to activate the PI3K/Akt signalling pathway 46

. Interestingly, this 44kDa truncated RXR has also been located in the mitochondrial matrix in which it exerts a transcriptional activity

47,48

, suggesting that RXR can shuttle between the cytoplasmic and nuclear compartments. In line with this, RXRα possess a nuclear localization signal and associates to importin , one of the cellular karyophilins binding the nuclear pore complex. When expressed as a GFP fusion protein, RXRα is mostly located in the nucleus but cytoplasmic RXR provides a piggyback transportation to the vitamin D receptor into the

Acknowledgements:

Work in our laboratory is supported by grants from the European Union (Xtranet), Région Nord-Pas de Calais, FEDER, and Fondation Coeur et Artères.