Sensitivity of muscle satellite cells to pollutants: an in vitro and in vivo comparative approach

Aquatic Toxicology 53 (2001) 247– 263 www.elsevier.com/locate/aquatox

Sensitivity of muscle satellite cells to pollutants: an in vitro and in vivo comparative approach B. Fauconneau *, G. Paboeuf Department of Hydrobiology and Wildlife, INRA, Campus de Beaulieu, 35042 Rennes Cedex, France

Abstract Muscle satellite cells from rainbow trout were exposed in vitro to increasing concentrations of different xenobiotics: copper, dichloroaniline, prochloraz, nonyl-phenol polyethexylate. Mortality and proliferation rate were measured by Hoechst binding and BrdU incorporation. Dose dependent effect of copper on survival and proliferation was observed with an EC50 at 100 mM. A dose dependent effect of nonyl-phenol diethoxylate was observed on survival with an EC50 at 100 mM. This was associated with a biphasic effect on proliferation rate observed both for nonyl-phenol di and poly-ethoxylate: a stimulation of proliferation at low concentration and an inhibition proliferation at large concentration. These effects were related to the inhibition of cells adhesion through the detergent capacity of nonyl-phenol polyethoxylate. The effects of prochloraze and dichloroaniline on cells mortality (EC 50 \500 mM) and proliferation rate (LOEC: 100 mM) were limited. Whole fish growth, muscle fibre size distribution and satellite cells survival and proliferation were measured on rainbow trout (60– 80 g BW) exposed to two concentrations of prochloraze (10 and 100 mg/ml) or nonyl phenol diethoxylate IGEPAL 210® (100 and 400 mg/ml) during 14 and 10 days exposure, respectively. Muscle fibre size distribution and satellite cells proliferation were affected by prochloraz exposure in vivo and this could be related to the alteration in fish feeding status. The exposure to IGEPAL 210® affected the number of satellite cells extracted and induce a biphasic effect on satellite cells proliferation similar to that observed in vivo. The combined exposure to IGEPAL 210® and prochloraze revealed additive effects of these two compounds. The in vivo and in vitro comparison demonstrated that in vitro satellite cells system could be used as a valuable tool to test the effects of pollutants. © 2001 Elsevier Science B.V. All rights reserved. Keywords: IGEPAL 210®; Satellite cells; Hypertrophy

1. Introduction



This work was supported by the EC Program Environment and Climate — Contract ENV4-CT96-0223 ‘Diagnostic Ecotoxicoly’ * Corresponding author. Tel.: +33-2-23485030; fax: + 332-23485034. E-mail address: [email protected] (B. Fauconneau).

Growth is generally the first integrative response of fish to environmental constraints. It is difficult however to dissociate between a direct effect of environmental constraints on growth process and an indirect effect related to alteration of other functions. This could be explored by

0166-445X/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 4 5 X ( 0 1 ) 0 0 1 7 0 - 9

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comparing the response of different tissue and cell type supporting growth and other functions (Segner et al., 2000). Muscle is one of the main tissue supporting growth process. Muscle growth is often used as an index of fish growth in response to governing environmental factors (oxygen, temperature, salinity …) (Houlihan et al., 1995; Fauconneau et al., 1997) and restraining environmental factors (pH, NH4, heavy metals) (Reid et al., 1995; Wilson et al., 1996). Very few studies are however available on the sensitivity of muscle growth to pollutants as compared to what have been made on the sensitivity of muscle locomotion and related criteria (Magnotti et al., 1997) to pollutants. Fish muscle growth is realised both by increasing the size of existing fibres (hypertrophy) and by increasing the number of fibres (hyperplasia) (Stickland, 1983; Koumans et al., 1993a). The two processes could be altered by pollutants. Both hyperplasia and hypertrophy processes required induction of muscle stem cells adjacent to existing fibres: myosatellite cells which proliferate and either fused with existing fibres (hypertrophy) or fused together to give new fibres (hyperplasia) (Fauconneau and Paboeuf, 2000a). These muscle stem cells are named satellite cells (Campion, 1984). Satellite cells are not easy to analyse in vivo due to their low density in muscle and due to the very few specific criteria available for identification of these totipotent stem cells (Koumans et al., 1991; Alfei et al., 1995; Johnston et al., 1999). It has thus been proposed to study these cells in vitro (Koumans et al., 1993b; Fauconneau and Paboeuf, 2000a). The characteristics of these cells and especially their proliferative capacity have been proved to be affected by intrinsic factors such as genetic origin and aging (Greenlee et al., 1995; Fauconneau and Paboeuf, 2000a) but also by extrinsic factors such as temperature and feeding activity (Matschak and Stickland, 1995; Fauconneau and Paboeuf, 2000b). The changes in the status of muscle satellite cells under environmental constraints have never been assessed. Thus, satellite cells studied both in situ and in

vitro could provide a valuable tool to analyse the specific effect of pollutants on muscle hyperplasic and hypertrophic growth. For each function (ion, water and gas exchange, detoxification, reproduction) and corresponding tissues (skin, gill, kidney, liver, gonad) affected by pollutants, the response of specific cells could also be analysed. It is however especially important to dissociate general cell responses to pollutants and specific effect on each of these cells system. The potential of applying in vitro cells systems in ecotoxicology by investigating different tissue and cell types responses and in vitro/ in vivo comparisons was thus explored in an European project (Segner et al., 2000). The aim of this work was to analysed the direct effect of different classes of pollutants: pesticides, heavy metals (copper), herbicides (dichloroaniline), fungicide (prochloraz) or additives detergents (nonyl-phenol poly ethoxylate) on muscle satellite cells in vitro. The chemicals studied were the reference compounds used in the different in vitro cell systems (Segner et al., 2000). The specific sensitivity of satellite cells to a pollutant observed in vitro was also tested in vivo by the effects of chronic exposure to selected compounds (prochloraz, nonyl-phenol poly ethoxylate) on muscle integrity and growth. 2. Material and methods

2.1. Animals and rearing conditions Fish of 1 –3 g body weight were used for the in vitro analysis of the effects of pollutants (see Table 1). Fish were adapted to experimental facilities 1 month before experiment and they were fed ad libitum during this period. A sample of 20–25 fish was weighed every week. Then fish were sampled for cell extraction and weighed individually. Depending on body weight, 40– 60 fish were sampled to obtain 20 g of white muscle. Fish of 60–80 g body weight were used in the in vivo chronic exposure to pollutants (see Table 2). In each experiment, groups of 30 fish were constituted and distributed randomly in 24× 160 l water ponds. The water in these tanks was

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aerated by bubbling of liquid oxygen and it was renewed by batch (half of water each day). Fish were fed at 1.5%.d − 1 manually in two meals and they were weighed every week. Fish were adapted during at least 15 days in these tanks before exposure to pollutants.

2.2. Cells extraction All the procedure described was realised at 15°C and cells were extracted using the procedure described by Fauconneau and Paboeuf (2000b). The same procedure was applied in small fish and in large fish. Fish were sampled, weighed, killed by a sharp blow on the head, immersed for 30 s in 70% ethanol. The skin was removed, the red superficial muscle was scrapped off and the dorsal white muscle was dissected carefully. Samples were immersed in basal medium on-ice: Dulbeco’s Modified Eagle Medium DMEM, NaHCO3 9 mM, HEPES 20 mM, pH 7.4, Posm 300 mOsm/kg supplemented with 15% Horse Serum. In the basal medium was added an antibiotic cocktail (penicillin 100 U/ml, streptomycin 100 mg/ml, fongizone 0.25 mg/ml, gentamycine 75 mg/ ml). Samples of muscle were finely cut, centrifuged (300×g, 5 min) and the pellet was washed two times with the basal medium without serum. The muscle tissue was then digested by collagenase (Type Ia Sigma 0.2% solution in DMEM) during 1 h at 18°C, then centrifuged (300×g, 5 min) and the pellet was washed with basal medium and re-suspended in 20 ml basal medium. The cells were then submitted to mechanical dissociation (five triturations through a 1.4 mm× 100 mm needle) and centrifuged (300×g, 20 min).

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The cells were then submitted two times to trypsin digestion (1:250 trypsin Sigma 0.1% solution in DMEM) during 20 min at 18°C, then centrifuged (300× g, 5 min) and the two supernatant were combined, diluted (1 v/1 v) in cold basal medium supplemented with 15% Horse Serum and centrifuged (300× g, 20 min, 4°C). The supernatant was re-suspended in 20 ml basal medium and the suspended cells were then submitted to mechanical dissociation (ten triturations through a 1.4 mm× 100 mm needle) and centrifuged (300× g, 20 min). The suspension was filtered successively on 100 mm and 50 mm nylon screen and centrifuged (300× g, 20 min, 4°C). The cells were re-suspended in the basal medium supplemented with 10% foetal calf serum to reach a final concentration of 1.5× 106 cells/ml. The yield of extraction obtained on the cells extracted were in the range of what was usually observed (2–4× 106 cells/g muscle, see Table 1).

2.3. Cell culture The crude cell suspension was enriched in satellite cells using their higher affinity for laminin. For this purpose plastic culture plates or glass cover slips (1.1 cm2) were treated successively with polyL-lysine 16 mg cm − 2 (Sigma MW\ 300 000, solution 100 mg ml − 1 in distilled water, 2 h 30 min at 12°C) and laminin 16 2 mg cm − 2 (L 2020 Sigma, 20 mg ml − 1 in DMEM, 24 h at 18°C). The cells were seeded on laminin treated plates at a concentration of 150 000–200 000 cells cm − 2 (see Table 1 depending on experiment) and left for 25 min at 18°C. The supernatant was then removed, the cells were washed gently two times with DMEM

Table 1 Characteristics of fish and satellite cells used for the in vitro experiments Experiments

Number of fish

Size of fish

CuSO4 CuCl2 Dichloro-aniline Prochloraze IGEPAL 720® IGEPAL 210®

120 26 21 56 39 56

0.8 2.8 3.0 2.8 2.4 2.8

g 90.2 g 90.3 g 90.4 g 9 0.4 g 90.3 g 9 0.4

g g g g g g

Extraction yield

Seeding density

2.5×106/g 3.7×106/g 2.3×106/g

1.5×105/cm2 1.5×105/cm2 2.0×105/cm2 1.5×105/cm2 2.0×105/cm2 1.5×105/cm2

1.5×106/g

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Table 2 Characteristics of fish used for muscle fibre analysis and characteristics of satellite cells extracted in the in vivo experiments Experiments

Number of fish

Size of fish

Extraction yield

Seeding density

Prochloraze+IGEPAL 210® IGEPAL 210®

10/treatment 10/treatment

58 g 98 g 76 g 9 11 g

0.7×106/g 0.7×106/g

1.5×105/cm2 1.5×105/cm2

then cultured at 18°C in the basal medium supplemented with 10% foetal calf serum. The medium was renewed every day in proliferation studies. The culture is characterised by different steps but only the initial period of proliferation was taken into account.

2.4. Proliferation analysis in 6itro: BrdU incorporation For proliferation studies, a solution of BrdU (Amersham): 10 mmol l − 1 in culture medium was added in the wells. Incorporation of BrdU from the culture medium was then measured during 24 h or 48 h periods. At the end of this period, the glass coverslips were immersed in cold (−20°C) ethanol and stored until analysis. The glass coverslips were washed in PBS 10% (phosphate buffer with 0.2% BSA), then incubated with a first monoclonal antibody (anti-BrdU clone BMC 9318 Boehringer), washed in PBS 10% pH 7.4, incubated with a second antibody (anti-mouse coupled with FITC 480 nm/535 nm) and washed in PBS 10%. Finally the glass coverslips were incubated in a Hoechst 33 258 (Molecular probes) dye solution for the revelation (355 nm/450 nm) of total cell nuclei. In vitro labelled fluorescent nuclei were counted on two representative areas (2× 0.0625 mm2) of the whole culture glass coverslips (\100 nuclei in each area). The total number of nuclei was assessed using Hoechst revelation and the proliferative nuclei was assessed using BrdU incorporation. A proliferative index was calculated as the mean ratio: proliferative nuclei/total nuclei in the two selected areas and expressed as % d − 1.

2.5. Muscle sampling and treatments for histology Fish of the in vivo experiments were sampled at

the end of the experiment for muscle histology (12 fish sampled in each treatment, four fish in each replicate) but also for satellite cells extraction (6 fish sampled in each treatment, two fish in each replicate) as described in cell extraction chapter. Muscle samples were collected in the caudal part of the fish between anal fin and adipose fin. The samples were fixed in Carnoy fixative (methanol/ dichloromethane/acetic acid 6/3/1 v), dehydrated and embedded in paraffin. Thin section (10 mm) were realised and treated with trichrome solution (sirius red, fast green). These sections were observed with a microscope at 20 or 40× and quantitative measurements (fibre area and fibre density) were made using an image analysis system (OPTIMAS Imasys, France).

2.6. In 6itro effect of pollutants Three different concentrations were tested for each pollutant (Table 3) depending on the toxicity range of the pollutant. These ranges were selected to elecit a clear toxic effects on fish cells at the maximum concentration (preliminary trials of J.P. Cravedi and M. Loir, see present issue). The only exception is dichloroaniline: 100% mortality was observed at 1000 mM in alternative cells models. Thus it was decided to utilise a lower maximum concentration (500 mM). Each concentration was tested in triplicates. Most of the pollutants were dissolved in water. For some of these pollutants a precipitate was observed for the large dose (dichloroaniline). Some other pollutants were however only soluble in DMSO. Although the concentration of DMSO used was low (0.05%), its effect on survival and proliferation of satellite cells was tested. DMSO had no effect on cell survival. It significantly stimulated initial satellite cells proliferation (Day 2) but had no effect on stimulated proliferation (Day

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4 and Day 6). Nonyl-phenol tested as a metabolite of IGEPAL 210® and IGEPAL 720® and oestradiol tested as a reference were only soluble in ethanol solution. Although the concentration of ethanol used was also low (0.1%), its effect on survival and proliferation of satellite cells was tested. Ethanol had no effect on cell survival but it significantly stimulated also initial satellite cells proliferation (Day 2). The effect of pollutants were tested on basal and stimulated proliferation of satellite cells. The pollutants were added after the enrichment step and were applied during all the culture. Proliferation was assessed by incorporation of BrdU during a 48 h period up to 6–8 days of culture depending on the intensity of the effect of the pollutant tested.

2.7. In 6i6o effects of pollutants In a first experiment the fish were exposed for 14 days to Prochloraz and to a combination of Prochloraze+IGEPAL 210®. In a second experiment, fish were exposed during 10 days only to IGEPAL 210® tested either in tap water and in river water (Seiche river 35 France). In these two experiments, each treatment was tested in triplicate. The pollutants were administrated regularly in the tank by peristaltic pumps in order to reach final concentrations mentioned in Table 4 (Loir et al., this issue).

2.8. Statistical analysis The mean and standard deviation of proliferation rate was calculated for each treatment tested.

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In each experiment the effect of the pollutant was tested using a one way analysis of variance. The differences between two doses of pollutant were then tested using a Student t-test. The difference in the mean percentile of each class of the muscle fibre size distribution were analysed using a Student t-test.

3. Results

3.1. Effect of copper on satellite cells sur6i6al and proliferation in 6itro Survival and Cu exposure was dose dependent: 1000 mM caused 100% mortality within 1 day (Fig. 1(a)), 100 mM killed all the cells at day 4 and 10 mM started to significantly affect survival at 6 days of culture. Initial proliferation of cells was not affected by copper (Fig. 1(b)). However after exposure to 10 mM during 2–4 days, proliferation decreased significantly. Proliferation was thus affected before any effect on cell survival.

3.2. Effect of dichloroaniline in 6itro Dichloroaniline had few toxic effect on satellite cells (Fig. 2(a–b)) as mortality was not modified (Fig. 2(a)) and proliferation was only affected at the higher concentration (500 mM) where dichloroaniline almost but completely inhibited the stimulated proliferation (day 4 and 6) but not the initial proliferation (day 2), Fig. 2(b)).

Table 3 Concentration of the different pollutants tested in vitro on muscle satellite cells extracted from 1 to 3 g body weight rainbow trout Compounds tested in vitro

Concentrations tested

Observations

Cu: CuSO4 and CuCl2

10 mM

100 mM

1000 mM

Dichloro-aniline Prochloraze IGEPAL 720® IGEPAL 210® 4-nonyl phenol 17ß Estradiol

10 mM 1 mM 1 mM 1 mM 1 mM 1 nM

100 mM 10 mM 10 mM 10 mM 10 mM 10 nM

500 mM 100 mM 100 mM

O2 consumption Protein synthesis Tested in DMSO 0.050/0 Tested in DMSO 0.05% Tested in DMSO 0.05% Tested in ethanol 0.10% Tested in ethanol 0.10%

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Table 4 Concentration of pollutants tested in vivo during 10–14 days on 60–80 g body weight rainbow trout Compounds tested in vivo

Concentrations tested

Prochloraze Prochloraze+IGEPAL 210®

10 mg/ml

100 mg/ml

IGEPAL 210®

100 mg/ml

400 mg/ml

3.3. Effect of prochloraz in 6itro Only 100 mM Prochloraz had a significant effect on cells survival (Fig. 3(a)). At this concentration, the effect was however still limited. At intermediate concentration (10 mM) Procholoraz significantly decreased survival only after few days of culture (at day 6). The higher concentration of prochloraz (100 mM) had no effect on initial proliferation measured at day 2 (Fig. 3(b) but significantly reduced stimulated proliferation at day 4 and inhibited proliferation at day 6. The other concentrations had no effect on satellite cells proliferation.

100 mg/ml Prochloraz 33 mg/ml IGEPAL

was significantly stimulated at 1 mM IGEPAL 720® after 2 days of culture but not after 4 days of culture (Fig. 5(b)). At 10 mM, satellite cell proliferation was significantly depressed and it is completely inhib-

3.4. Effect of nonyl phenol diethoxylate (IGEPAL 210 ®) in 6itro Nonyl phenol diethoxylate IGEPAL 210® had a significant effect on survival at 100 mM (Fig. 4(a)). At 10 mM, survival was significantly lower only at day 2. At day 2 during the basal initial proliferation period, the response to nony phenol diethoxylate demonstrated a clear dose response (Fig. 4(b)). At day 4 and day 6 the response was less clear although the higher concentration of IGEPAL 210®, 100 mM significantly depressed proliferation of satellite cells. These effects were not associated with any changes in morphologies of cells as they tend to lose their adhesion capacities and cells remains were found floating in the culture medium.

3.5. Effect of nonylphenolpolyethoxylate (IGEPAL 720 ®) in 6itro IGEPAL 720® had no effect on cell survival at 1 and 10 mM (Fig. 5(a)). Satellite cell proliferation

Fig. 1. Effect of in vitro copper exposure (10, 100, 1000 mM) on white muscle satellite cells: (a) number (nb/cm2); and (b) proliferation (% BrdU positive cells 48 h-1) in 2 – 4 g BW juvenile rainbow trout (see Tables 1 and 2 for more details). Mean + Standard Deviation (n =3).

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nonylphenol-polyethoxylate but no significant effect were observed neither on survival nor on satellite cells proliferation (Fig. 6(a–b)). 17-ßoestradiol was tested on satellite cells in a range of concentration (1– 10 nM), 1000 times lower as those tested for nonyl phenol and nonyl phenol polyexthoxylate and dietethoxylate. 17 ß oestra-

Fig. 2. Effect of in vitro dichloro-aniline exposure (10, 100, 500 mM) on white muscle satellite cells: (a) number (nb/cm2); and (b) proliferation (% BrdU positive cells 48 h − 1) in 2 –4 g BW juvenile rainbow trout. Dichloroaniline was dissolved in a 0.05% DMSO solution of the culture medium (see Tables 1 and 3 for more details). Mean +Standard Deviation (n= 3).

ited after 4 days of culture. Thus the response to IGEPAL 720® is not just dose dependent.

3.6. Effect of nonyl phenol and 17 ß estradiol in 6itro Nonyl-phenol a metabolite of nonyl-phenolpolyethoxylate and nonyl-phenol-die´ thoxylate was tested in the same range of concentration as for

Fig. 3. Effect of in vitro procholoraz exposure (1, 10, 100 mM) on white muscle satellite cells: (a) number (nb/cm2); and (b) proliferation (% BrdU positive cells 48 h − 1) in 2– 4 g BW juvenile rainbow trout. Procholoraz was dissolved in a 0.05% DMSO solution of the culture medium (see Tables 1 and 3 for more details). Mean +Standard Deviation (n = 3).

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compared to control fish. Treated fish tended however to loss body weight during the 14 days experimental period compared to control fish which have the same body weight at the end of the experiment than the initial body weight. This was especially true for the group of fish exposed to both prochloraze (100 mg/ml) and IGEPAL 210 (33 mg/ml) ( − 10% compared to initial body weight). The size (area) of muscle fibres were measured in control and treated fish ( 100 fibres/fish) on transverse section of white muscle. The complete distribution of the size of white muscle fibres was modified by the treatments (Fig. 9(a)). Both a

Fig. 4. Effect of in vitro IGEPAL 210® exposure (1, 10, 100 mM) on white muscle satellite cells: (a) number (nb/cm2); and (b) proliferation (% BrdU positive cells 48 h − 1) in 2–4 g BW juvenile rainbow trout. IGEPAL 210® was dissolved in a 0.05% DMSO solution of the culture medium (see Tables 1 and 3 for more details). Mean + Standard Deviation (n= 3).

diol had no significant effects on survival and proliferation at these concentrations (Fig. 7(a– b)).

3.7. Effect of prochloraz and combined effect of prochloraz and IGEPAL 210 ® in 6i6o (experiment 1) After 14 days of exposure to prochloraze alone or combined with IGEPAL 210®, there was no significant differences in body weight (Fig. 8(a))

Fig. 5. Effect of in vitro IGEPAL 720® (1, 10 mM) on white muscle satellite cells: (a) number (nb/cm2); and (b) proliferation (% BrdU positive cells 48 h − 1) in 2– 4 g BW juvenile rainbow trout (see Tables 1 and 2 for more details). Mean + Standard Deviation (n =3).

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fish exposed to prochloraze and IGEPAL 210®. The exposure in vivo to different pollutants affected the survival of satellite cells in vitro (Fig. 10(a)). The cells from fish treated with prochloraz only tended to have a lower survival than the control but the differences were only significant at day 4, 6 and 8 at the lower concentration tested (10 mg/l) and at day 8 at the higher concentration (100 mg/ml). In the fish treated with both prochloraz and IGEPAL 210®, a significant decrease in cells number was observed during all the culture compared to control fish and fish treated only with prochloraz.

Fig. 6. Effect of in vitro nonyl-phenol exposure (1, 10 mM) on white muscle satellite cells: (a) number (nb/cm2); and (b) proliferation (% BrdU positive cells 48 h − 1) in 2–4 g BW juvenile rainbow trout. Nonyl phenol was dissolved in a 0.1% ethanol solution of the culture medium (see Tables 1 and 3 for more details). Mean +Standard Deviation (n= 3).

decrease in the proportion of the small fibres population and a decrease in the mean size of white muscle fibre were observed (Fig. 9(b)) was observed in treated fish compared to the control. The mean diameter of fibres was lower in fish exposed to prochloraze in a dose dependent manner compared to control fish. The fish exposed to both prochloraze and IGEPAL 210® had the lowest mean muscle fibre diameter (Fig. 9(b)). The number of satellite cells extracted from white muscle was only measured on the whole batch for each treatment, it is thus not possible to conclude on a significant effect of the treatments on this parameter. Prochloraz tended to increase the yield of extraction (Fig. 8(b)). On the opposite the number of satellite cells extracted was lower in

Fig. 7. Effect of in vitro 17-b-estradiol exposure (1, 10 nM) on white muscle satellite cells: (a) number (nb/cm2); and (b) proliferation (% BrdU positive cells 48 h − 1) in 2– 4 g BW juvenile rainbow trout. 17-b-estradiol was dissolved in a 100 mg/ml ethanol solution of the culture medium (see Tables 1 and 3 for more details). Mean +Standard Deviation (n =3).

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3.8. Effect of IGEPAL 210 ® in 6i6o (experiment 2)

Fig. 8. Effect of in vivo procholoraz (10, 100 mg/l) and procholoraz+ IGEPAL 210® (100 and 33 mg/l, respectively) 14 days exposure on: (a) body weight (g) (mean + standard deviation n=10); and (b) white muscle satellite cells extraction yield (nb g − 1 muscle) in 50 g BW juvenile rainbow trout (see Tables 2 and 4 for more details).

After a 10 days exposure to IGEPAL 210®, the body weight of fish exposed to high concentration of IGEPAL 210® (400 mg/ml) was lower than in the other group (Fig. 11(a)). The differences between the other groups were not significant. The size (area) of muscle fibres could not be measured in control and treated fish in this experiment as the transverse section were in very bad state probably related to the feeding state of fish. The treatment in vivo with IGEPAL 210® was associated with a decrease in the number of satellite cells extracted (Fig. 11(b)). The effect seems to be more important at large dose but as there was no replicate (cells extracted from a pool of muscle in each treatment) it is not possible to conclude on the significance of such an effect. The different treatments applied had no effect on survival of fish satellite cells in vitro (Fig. 12(a)). The in vitro proliferation of satellite cells from fish exposed to the lower concentration of IGEPAL 210® in vivo was significantly higher than the control at day 2 and day 6 (Fig. 12(b)). The in vitro proliferation rate of satellite cells from fish exposed to the high concentration of IGEPAL 210® (400 mg/ml) was significantly lower than the control and the 100 mg/ml treatment (Fig. 12(b)). No differences in the two groups treated with the higher concentration of IGEPAL 210® were observed.

4. Discussion

4.1. Effect of pollutants on satellite cells features in 6itro The proliferation of satellite cells was significantly lower in fish exposed to procholoraz at day 4 and 6 (10 mg/ml) and day 6 (100 mg/ml) but not at day 2 and at day 8. The proliferation was significantly lower in fish exposed to both prochloraz and IGEPAL 210® at days 2, 4 and 6 compared to control fish. The proliferation of satellite cells was significantly lower in fish exposed to both prochloraz and IGEPAL 210® than that in fish exposed only to prochloraz at day 4.

The main effects of the different pollutants tested in vitro could be compared on Table 5. Satellite cells extracted from muscle and cultured in vitro could be affected by the toxicity of the different xenobiotics tested and this was measured as for other cells types by cells mortality. Satellite cells survival was greatly affected by low concentrations of copper compared to other compounds tested. Significant effects were observed at the low

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concentration tested (10 mM after 6 days of culture) for survival and proliferation of satellite. Some response were also observed for protein synthesis of myotubes (8 days of culture) but at largely higher concentration (1000 mM) (Smith, Houlihan and Fauconneau personal communication). Copper is highly toxic for all cell types and alteration of feeding and growth have been observed in fish after exposure to sublethal concentration (Doeboeck et al., 1998; Smith et al., 2000). Thus, the response of muscle satellite cells is probably a non specific toxic response. Satellite

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cells actively involved in cell cycles and differentiation could be especially sensitive. The Lower Observed Effect Concentration (LOEC), i.e. 10 mM is however far higher than the concentration of copper observed in polluted area and an in vitro specific effect on muscle growth could not be emphasized. In vivo myosatellite cells are included in basa lamina of existing fibres (Campion, 1984). It is supposed that satellite cells are induced in vivo for myogenesis by disruption of basa lamina either by microstretch (exercise) (Darr and Schultz, 1987)

Fig. 9. Effect of in vivo procholoraz (10, 100 mg/l) and procholoraz +IGEPAL 210® (100 and 33 mg/l, respectively) 14 days exposure on: (a) white muscle fibre diameter distribution; and (b) mean white muscle fibre width and equivalent-diameter in 50 g BW juvenile rainbow trout (see Tables 2 and 4 for more details). Mean + Standard Deviation (n = 5).

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Nonyl phenol poly-ethoxylate is included in pesticide and herbicide solution for its known detergent effect which is directly related to the length of the ethoxylate chain. The negative effect of IGEPAL nonyl-phenol polyethoxylate could thus be related to an inhibition of cells adhesion through its detergent action. Satellite cells realised in vitro a complete myogenesis process: proliferation, differentiation of

Fig. 10. Effect of in vivo procholoraz (10, 100 mg/l) and procholoraz+ IGEPAL 210® (100 and 33 mg/l, respectively) 14 days exposure on white muscle satellite cells: (a) number (nb/cm2); and (b) proliferation (% BrdU positive cells 48 h − 1) in 50 g BW juvenile rainbow trout (see Tables 2 and 4 for more details). Mean + Standard Deviation (n= 3).

or biochemical processes (paracrine control). When isolated from muscle tissue, satellite cells in vitro adhere to their specific substrate (component of basa lamina) and start the myogenesis process. Furthermore, freshly differentiated satellite cells have the capacity to migrate. Migration involves specific attachment and detachment from components of extracellular matrix (laminin, integrin). Thus, the adhesion capacity of satellite cells could be one of the specific target of xenobiotics. This was probably observed with the high concentration of IGEPAL 210® which affect negatively both cells number and proliferation. The same effects were observed at lower dose with IGEPAL720® but only for cells proliferation. The effects of nonyl phenol-polyethoxylate and diethoxylate were specific as nonyl-phenol the final metabolite of nonyl-phenol polyethoxylate (Jobling et al., 1996) and oestrogen had no effect on satellite cells mortality and proliferation.

Fig. 11. Effect of in vivo IGEPAL 210® (100, 400 mg/l in tap water and 400 mg/l in river water) 10 days exposure on: (a) body weight (g) (mean +standard deviation, n =10); and (b) white muscle satellite cells extraction yield (nb g − 1 muscle) in 50 g BW juvenile rainbow trout (see Tables 2 and 4 for more details).

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polyethoxylate. At very low concentration a positive effect of IGEPAL 720® on satellite cells proliferation was observed without any significant effect on cells mortality. The effect of IGEPAL 210® was similar than that of IGEPAL 720® but the stimulation of proliferation was observed at higher concentration. The same response was observed for ethanol and DMSO when tested in the control medium at very low concentrations. It could thus be emphasized that IGEPAL 210® and IGEPAL 720® would inhibit partially adhesion of cells at low doses and this will indirectly inhibit differentiation of satellite cells and in turn stimulate proliferation. The bimodal effects of nonylphenol polyethoxylate could only be demonstrated using the in vitro satellite cells culture system. The satellite cells of fish generally expressed in vitro a ‘basal’ proliferation (days 0–2) which depends on the status of cells in vivo and a ‘stimulated’ proliferation (days 2–6) induced by growth factors of the cultured medium are generally obTable 5 In vitro effects of pollutants on myosatellite cells from 1 to 3 g body weight rainbow trout frya Fig. 12. Effect of in vivo IGEPAL 210® (100, 400 mg/l in tap water and 400 mg/l in river water) 10 days exposure on white muscle satellite cells: (a) number (nb/cm2); and (b) proliferation (% BrdU positive cells 48 h − 1) in 50 g BW juvenile rainbow trout (see Tables 2 and 4 for more details). Mean + Standard Deviation (n = 3).

cells, fusion in multinucleated cells, differentiation in myotubes. In many mammals, a balance between proliferation and differentiation of satellite cells could be observed in vitro by a clear separation between a proliferative period and a differentiation period which have to be induce by specific factors (Olson, 1992). In fish, differentiation started in some cells as soon as cells are seeded when other cells still proliferate and thus there is an overlapping of the proliferation and differentiation processes (Fauconneau and Paboeuf, 2000a). Exposure to xenobiotics could however specifically revealed the balance between proliferation and differentiation as it was illustrated by the interesting response to nonylphenol

Compound

Cells survival

Cells Proliferation

Cu

10 (LOEC) 100 (EC50)

Dichloroaniline

No effect

Prochloraze

10 (LOEC) 100 (EC50)

Nonyl Phenol Polyethoxylate IGEPAL 720®

No effect up to 100 mM

10 (LOEC) Time×dose response Effect at large concentration 500 mM 10–100 (LOEC) Inhibition at 100 mM 10 (LOEC)

Nonyl Phenol Diethoxylate IGEPAL 210®

10 (LOEC)

100 (EC50) Nonyl Phenol Estradiol 17ß a

No effect No effect

LOEC and EC50 values in mM.

Inhibition at 10 mM 100 (LOEC)

Inhibition at 100 mM No effect No effect

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served (Fauconneau and Paboeuf, 2000a). Xenobiotics would affect rather stimulated proliferation than basal proliferation in vitro and this was observed for the response to dichloroaniline and prochloraz especially at the high dose tested (500 and 100 mmol/l, respectively). The effect of in vivo factors on satellite cells status and thus on basal proliferation could be illustrated by the comparison between the culture realised for the in vitro exposure and that realised for the in vivo exposure to pollutants. The basal proliferation of satellite cells from small (2– 3 g BW) well fed fish were largely higher than that of satellite cells from large (50– 60 g) and restrained fish (15 – 25% 48 h versus 2– 3%, respectively). This could be related both to fish size and feeding status (Fauconneau and Paboeuf, 2000a,b).

4.2. Effects of pollutants in 6i6o 4.2.1. Fish and muscle growth The body weight of fish sampled in the first experiment was not significantly affected by the treatments with pollutants in vivo. This is probably related to the short duration of the experiment (10 days). The group treated with IGEPAL 210® (33 mg/ml) in the first experiment tended (P B0.1) however to have the lowest body weight. In the second experiment, the two groups treated during 14 days with the large dose of IGEPAL 210® (400 mg/ml) have significant lower body weight than the control. These results suggest that nonyl-phenol polyethoxylate would have an effect on fish growth at concentration related to what could be observed in the environment (Blackburn et al., 1999). Muscle growth process resulted on an hypertrophy of existing muscle fibres and the building of new fibres. These two processes could be directly analysed in the distribution of muscle fibres size (Stickland, 1983; Koumans et al., 1993a). In the first experiment, specific changes in the distribution of muscle fibres were observed with a disappearance of the small diameter fibre population and a decrease in the mean size of the remaining fibres in the treated groups compared with the control group. This demonstrate that both hyperplasic and hypertrophic processes were affected by exposure to pollutants. Changes in muscle growth processes

are probably much more rapid than the changes in fish body weight for which only trends were observed. These changes in distribution of white muscle fibre were however very similar to what is observed with feed restriction (Kiessling et al., 1991) and it could thus be proposed that the different pollutants have an effect on fish growth through food restriction. The changes in muscle fibre distribution were related with the degree of pollutants exposure: prochloraz (10 mg/ml) Bprochloraz (100 mg/ml) B prochloraz (100 mg/ml) + IGEPAL® (33 mg/ml) These changes suggest that muscle growth could be affected in a dose dependent manner by pollutants such as prochloraze and that the effects of pollutants on muscle growth could be additive. Unfortunately, the muscle samples of the second experiment could not be analysed and it is this not possible to conclude on specific effects of prochloraz or nonyl-phenol polyethoxylate on fibre size distribution and thus muscle growth.

4.2.2. Satellite cells In this work it could simply tested directly if the effects observed on satellite cells exposed in vitro to pollutants are also observed when fish are exposed in vivo to pollutants. Before any comparison, it could be mentionned that the yield of extraction of satellite cells (Tables 1 and 2), the survival of satellite cells in vitro and the basal proliferation rate (day 2) observed in the two in vivo experiments were largely lower than what was observed in the different in vitro experiments. These differences were directly related to the decrease in the number and activity of satellite cells with ageing/size of fish (Alfei et al., 1995; Koumans et al., 1993b; Fauconneau and Paboeuf, 2000a) as fish used for the in vivo experiments have a higher body weight (60–80 g) than those used for the in vitro experiments (1–3 g). However some of these differences such as those observed for satellite cells proliferation could also be related to differences in feeding status as fish in the in vivo experiment were restrained compared to fish in the in vitro experiment which were fed ad libitum. The response of satellite cells to xenobiotics exposure in vivo are summarised in Table 6.

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Table 6 Growth and myosatellite cells survival and proliferation in vitro in rainbow trout (60–80 g BW) exposed during 14 and 10 days, respectively to prochloraze (10 and 100 mg/ml) and IGEPAL 210® (100 and 400 mg/ml.

Prochloraze Prochloraze+Nonyl Phenol Diethoxylate IGEPAL 210® Nonyl Phenol Diethoxylate IGEPAL 210®

Weight of fish

Cells extracted

Cells Survival

No effect (−) No effect

P

(])

o

o

o

No effect

o

o at high dose

P at low dose No effect or o at high dose

Differences in yield of extraction were observed between the different treatments however the extraction of cells were not realised in replicates so that no statistical analysis could be realised on this parameter. The yield of extraction was however higher in the two prochloraze group compared to control and other treated group. This could be related directly to feeding status as it has been shown that food restriction is associated with a higher yield of extraction of satellite cells when compared to normal ad libitum feeding (Fauconneau and Paboeuf, 2000a,b). It is supposed to result on the relative increase in the density of satellite cells during fasting but also to an increase resistance of satellite cells to extraction process. This observation together with changes in muscle fibres distribution demonstrate that prochloraz treatment induced an alteration in muscle growth through a reduction in food intake. It is not known if it is mediated by a direct effect on voluntary feed intake. The mortality of satellite cells in vitro was affected by the treatments in vivo with prochloraze (first experiment) as it was observed in the in vitro experiments. The proliferation capacities of satellite cells in vitro were negatively affected by prochloraz exposure in vivo at 10 and 100 mg/ml and the same was also observed in the in vitro experiment. It means that the intrinsic capacities of satellite cells were affected by prochloraz exposure in vivo and this induce differences in muscle growth. The effects of prochloraze on fish muscle growth in vivo could be thus well predicted from the in vitro experiments.

Cells Proliferation

The survival of satellite cells in vitro was not affected by the in vivo exposure to nonyl-phenol polyethoxylate (100 and 400 mg/ml) but was affected in a dose dependent manner by the direct exposure in vitro. The effects of IGEPAL 210® observed in vitro were supposed to be related to a direct detergent effect on the inhibition of cells adhesion and this is consistent with the fact that such an effect could not be observed on cells only exposed in vivo to IGEPAL 210®. The effect observed in vitro on cells survival could however be related to the fact that in the two in vivo experiments the yield of satellite cells extraction in fish treated with IGEPAL 210 ® was lower than that of the control. This difference was opposite to that generally induce by feed restriction which is associated with an increase in the number of satellite cells extracted from muscle (Fauconneau and Paboeuf, 2000a,b). It could thus be proposed that nonyl-phenol specifically affect either the number of satellite cells in vivo or the sensitivity of satellite cells to extraction process. The underlying mechanisms could be similar in vivo and in vitro a inhibitory effect of the detergent action of nonyl-phenol polyethoxylate on adhesion of satellite cells. This is associated in vivo with differences in muscle growth processes and consequently in fish growth within a few days. The response of satellite cells proliferation to IGEPAL 210® exposure in vivo were similar to what was observed in the in vitro experiment both for the stimulation of proliferation at low concentration and for the inhibition of proliferation at high concentration. Thus the bimodal detergent effect of nonyl-phenol ethoxylate on satellite cells

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proliferation observed in vitro also occurred in vivo. It could be emphasised that nonyl-phenol polyethoxylate affect either the environment of satellite cells which are included in basa lamina (Koumans et al., 1993a; Johnston et al., 1999) or the membrane characteristics and receptors involved in the adhesion capacities of satellite cells (Greenlee et al., 1995; Fauconneau and Paboeuf, 2000a). These effects are consistent with the effect on cells survival in vitro and cell number in vivo. It is associated in vivo with differences in muscle growth processes and consequently in fish growth within a few days. These findings are really original and needs further investigations. A large significant combined effect of Prochloraz (100 mg/ml) and IGEPAL 210® (33 mg/ml) in vivo was observed on the survival of satellite cells and on the proliferation of satellite cells. The effects of these two compounds were found to be different in vitro and related to different mechanisms: a non specific toxic effect for prochloraz (Bach and Sengraoff, 1989) and a specific detergent effect for nonyl-phenol polyethoxylate. The cells whose environment was altered (adhesion capacities) were probably more sensitive to a toxic compounds. The additive effect of the two compounds in vivo on muscle growth could thus have been predicted from what was observed in vitro.

5. Conclusions The use of muscle satellite cells system to assess the effect of different pollutants on muscle growth is complementary to the use of other cells models. These cells have specific characteristics: stem cells, cells included in basa lamina, actively proliferative cells, balance between proliferation and differentiation that are important targets for the different pollutants tested. Satellite cells are sensitive to copper in vitro and this is consistent to what was observed in vivo for muscle growth and muscle protein synthesis (Smith et al., 2000). The responses to prochloraz and nonyl-phenol polyethoxylate in vitro have been found to be closely related to what was observed in vivo for satellite cells and muscle growth processes after exposure to these compounds. The

complex response to nonyl-phenol polyethoxylate observed in vitro was also observed in vivo. The in vitro muscle satellite cells system could thus be used as a feasible method in ecotoxicology. It could help to screen various compounds and identify the underlying mechanisms that could not be elucidated easily in vivo by testing various factors. Growth is however a cumulative process and such an in vitro cell system could thus only predict the effect of a specific compound on muscle growth but not its timing and its intensity. This is probably a limit for its use in ecological risk assessment. The responses of satellite cells in vitro (yield of extraction, survival, proliferation) to treatments applied on fish in vivo suggest that these cells could also be used as a valuable tool for diagnostic. References Alfei, L., Onali, A., Spano, L., Colombari, P.T., Altavista, P.L., de Vita, R., 1995. PCNA/Cyclin expression and BrdU uptake define proliferating myosatellite cells during hyperplastic muscle growth of fish (Cryprinus carpio L.). Eur. J. Histochem. 38, 151 – 162. Bach, J., Sengraoff, J., 1989. Effects of the fungicide prochloraz on xenobiotics metabolism in rainbow trout: in vivo induction. Xenbiotica 19, 1 – 9. Blackburn, M.A., Kirby, S.J., Waldock, M.J., 1999. Concentrations of alkylphenol poly ethoxylate entering UK estuaries. Ecotoxicol. Environ. Saf. 43, 213 – 221. Campion, D.R., 1984. The muscle satellite cells: A review. Int. Rev. Cytol. 87, 225 – 251. Darr, K.C., Schultz, E., 1987. Exercice induced satellite cell activation in growing and mature skeletal muscle. J. Appl. Physiol. 63, 1816 – 1821. Doeboeck, G., Vlaeminck, A., Blust, R., 1998. Effects of sublethal copper exposure on copper accumulation, food consumption, growth, energy stores and nucleic acid content in common carp. Arc. Env. Cont. Tox. 33, 415 – 422. Fauconneau, B., Andre, S., Chmaitilly, J., Le Bail, P.Y., Krieg, F., Kaushik, S.J., 1997. Control of skeletal muscle fibres and adipose cells in the flesh of rainbow trout. J. Fish Biol. 50, 296 – 314. Fauconneau, B., Paboeuf G., 2000a. Muscle satellite cells in fish. In: Johnston, I.A. (Ed.), Muscle Development and Growth in Fish, Physiology, vol. XVIII, Academic Press, New York, in press. Fauconneau, B., Paboeuf, G., 2000b. Effect of fasting and refeeding on satellite cells status in vitro of rainbow trout. Cell. Tissue Res. 301, 459 – 463. Greenlee, A.R., Kersten, C.A., Cloud, J.G., 1995. Effects of triploidy on rainbow trout myogenesis in vitro. J. Fish Biol. 46, 381 – 388.

B. Fauconneau, G. Paboeuf / Aquatic Toxicology 53 (2001) 247–263 Houlihan, D.F., Carter, C.G, McCarthy, I.D., 1995. Protein synthesis in fish. In: Hochachka, Mommsen (Eds.), Biochemistry and Molecular Biology of Fishes, vol. 4, Elsevier Science, pp. 191 – 220. Jobling, S., Shealan, D., Osborne, J.A., Matthiessen, P., Sumpter, J.P., 1996. Inhibition of testicular growth in rainbow trout (Oncorhynchus mykiss) exposed to estrogenic alkylphenolic chemicals. Env. Tox. Chem. 15, 194 – 202. Johnston, I.A., Strugnell, G., McCraken, M.L., Johnstone, R., 1999. Muscle growth and development in normal sex ratio and all-female diploid and triploid Atlantic salmon. J. Exp. Biol. 202, 1991 – 2016. Kiessling, A., Storebakken, T., Asgard, T., Kiessling, K.H., 1991. Changes in the structure and function of the epaxial muscle of rainbow trout (Oncorhynchus mykiss) in relation to ration and age: I Growth Dynamics. Aquaculture 93, 335 – 356. Koumans, J.T.M., Akster, H.A., Booms, G.H.R., Lemmens, C.J.J., Osse, J.W.M., 1991. Numbers of myosatellite cells in white axial muscle of growing fish Cyprinus carpio L. (Teleostei). Am. J. Anat. 192, 418 –424. Koumans, J.T.M., Akster, H.A., Booms, G.H.R., Osse, J.W., 1993a. Growth of carp (Cyprinus carpio) white axial muscle; hyperplasia and hypertrophy in relation to the myonucleus/sarcoplasm ratio and the occurrence of different subclasses of myogenic cells. J. Fish Biol. 43, 69 –80. Koumans, J.T.M., Akster, H.A., Booms, G.H.R., Osse, J.W., 1993b. Influence of fish size on proliferation and differentiation of cultured myosatellite cells of white axial muscle of carp (Cyprinus carpio L.). Differentiation 53, 1 –6.

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Magnotti, R.A. Jr., Zaino, J.P., Mc Connell, R.S., 1997. Pesticide-sensitive fish muscle cholinesterase. Comp. Biochem. Physiol. 108B, 187 – 194. Matschak, T.W., Stickland, N.C., 1995. The growth of Atlantic salmon (Salmo salar L.) myosatellite cells in culture at two different temperatures. Experientia 51, 260 – 266. Olson, E.N., 1992. Interplay between proliferation and differentiation within the myogenic lineage. Dev. Biol. 154, 261 – 272. Reid, S.D., Dockray, J.J., Linton, T.K., McDonald, D.G., Wood, C.M., 1995. Effect of a summer temperature regime representative of global warming scenario on growth and protein synthesis in hard water and soft water acclimated juvenile rainbow trout (Oncorhynchus mykiss). J. Therm. Biol. 20, 231 – 244. Segner, H, Chesne´ , C., Cravedi, J.P., Fauconneau, B., Houlihan, D.F., LeGac, F., Loir, M., Mothersill, C., Pa¨ rt, P., Valotaire, Y., Prunet, P., 2000. Cellular approaches for diagnostic effects assessment in ecotoxicology: introductory remarks to an EU-funded project. Aquat. Toxicol., this issue. Smith, R.W., Dowling, K., Jo¨ nsson, M., Sturm, A., Houlihan, D.F., 2000. Protein synthesis in rainbow trout following pollutant exposure: an evaluation of an investigative tool in sublethal toxicology, this issue. Stickland, N.C., 1983. Growth and development of muscle fibres in the rainbow trout (Salmo gairdneri ). J. Anat. 137, 323 – 333. Wilson, R.W., Wood, C.M., Houlihan, D.F., 1996. Growth and protein turnover during acclimation to acid and aluminium in juvenile rainbow trout (Oncorhnychus mykiss). Can. J. Fish Aquat. Sci. 53, 802 – 811.