Physiological morphological and metabolic changes in Tetrahymena pyriformis for the in vivo cytotoxicity assessment of metallic pollution: Impact on d-β-hydroxybutyrate dehydrogenase

Physiological, morphological and metabolic changes in Tetrahymena pyriformis for the in vivo cytotoxicity

assessment of metallic pollution: Impact on

D -b-hydroxybutyrate dehydrogenase

Driss Mountassif ^a, * , Mostafa Kabine ^a , Rachid Manar ^b , Noureddine Bourhim ^a , Zaina Zaroual ^c , Norbert Latruffe ^d , M’Hammed Saı¨d El Kebbaj ^a

a

Laboratoire de Biochimie et Biologie Mole´culaire, Faculte´ des Sciences, Universite´ Hassan II-Aı¨n Chock, km 8 route d’El Jadida BP. 5366 Casablanca, Morocco

b

Laboratoire de Biologie et Ecologie Animale, Faculte´ des Sciences, Universite´ Hassan II-Aı¨n Chock, km 8 route d’El Jadida BP. 5366 Casablanca, Morocco

c

Laboratoire d’Electrochimie et Chimie de l’Environnement, Faculte´ des Sciences, Universite´ Hassan II-Aı¨n Chock, km 8 route d’El Jadida BP. 5366 Casablanca, Morocco

d

LBMC (GDR-CNRS no. 2583), Faculte´ des Sciences, 6 Bd Gabriel, Universite´ de Bourgogne et Centre de Recherche INSERM, Faculte´ des Sciences, 21000 Dijon Cedex, France

Received 30 July 2006; received in revised form 19 November 2006; accepted 20 November 2006

Abstract

The individual cytotoxicity of cadmium chloride, iron sulphate and chromium nitrate has been investigated by using the freshwater ciliate Tetrahymena pyriformis. The metabolic enzymes and antioxidant defense biomarkers were assessed. The results obtained reveal that their metal salts have perturbed the physiology and morphology of T. pyriformis. Also, the biomarkers assessed were sensitive to the presence of metal salts and this sensitivity was metal salt and dose dependant. To estimate the impact of their metal salts on mitochondria, we studied their effects in vivo and in vitro on the

-b-hydroxybutyrate dehydrogenase (BDH) (EC 1.1.1.30) inner mitochondrial membrane enzyme. The results showed a high inhibition of BDH in terms of activity, protein expression and kinetic parameters.

# 2006 Elsevier Ltd. All rights reserved.

Keywords: Tetrahymena pyriformis; Cytotoxicity; Metal salts; Metabolic enzymes; Antioxidant biomarkers; Mitochondria;

-b- hydroxybutyrate dehydrogenase

This article is also available online at:

www.elsevier.com/locate/ecolind

Abbreviations: BDH,

-b-Hydroxybutyrate dehydrogenase;

-BOH,

-b-Hydroxybutyrate; DCIP, Dichloroindophenol; DTNB, Dithio- nitrobenzene; DTT, Dithiotreitol; EDTA, Ethylenediamine tetraacetic acid;

-G3P,

-glyceraldehyde-3-phosphate; KCN, Potassium cyanide;

MDA, Malondialdehyde; NAD(H), Nicotinamide adenine dinucleotide oxidized (reduced) forms; NADPH, Nicotinamide adenine dinucleotide phosphate reduced form; PMSF, Phenylmethylsulphonylfluoride

* Corresponding author.

1470-160X/$ – see front matter

2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.ecolind.2006.11.010

1. Introduction

Environmental pollutants have long-term effects on cellular development. Chronic exposure to pollutants via food and drinking water is a major human health concern. The transition metal has been widely used in industry. Toxic doses of transition metals are able to disturb the natural oxidation/

reduction balance in cells through various mechan- isms stemming from their own complex redox reactions, which perturb the cellular signaling and gene expression systems (Buzard and Kasprazak, 2000). Ultimately, there is a variety of toxic effects, including apoptosis and carcinogenesis (Kondo et al., 2000; Waalkes, 2000; Ye et al., 2000; Ishido and Kunimoto, 2001).

To evaluate the impact of metal salts on cell redox balance, we used as model system the NAD-dependent

D

-b-hydroxybutyrate dehydrogenase (EC. 1.1.1.30) (BDH), a ketone body converting enzyme.

BDH was largely studied in several organisms, especially in rat liver (Latruffe and Gaudemer, 1974), beef heart (Sekuzu et al., 1963; Nielsen et al., 1973), or Rhodopseudomonas spheroides (Bergmeyer et al., 1967). It catalyses the reversible oxidation of b- hydroxybutyrate to acetoacetate in presence of NAD (H) as cofactor, according to an ordered bi bi mechanism where the coenzyme binds first to the enzyme catalytic site (Nielsen et al., 1973; Latruffe and Gaudemer, 1974). While the enzyme is soluble in bacteria (Nielsen et al., 1973), in contrast, in eukaryotes it is embedded in inner mitochondrial membrane (Latruffe and Gaudemer, 1974).

The freshwater ciliate Tetrahymena pyriformis is an organism of choice on cytotoxicity studies (Nilsson, 1988). Its short generation time compared to invertebrates (Viarengo et al., 1990; Livingstone, 1992) and the fact that it can be grown quickly in culture are especially advantageous for studying the action of xenobiotics on several generations of cells.

This ciliate has been used to determine the effects of different xenobiotics (Bamdad et al., 1995).

The purpose of this study is to provide the impact of metallic pollution by cadmium, chromium and iron on the T. pyriformis cellular antioxidants and metabolic systems especially their effects on the

-b-hydro- xybutyrate dehydrogenase in terms of activity, protein expression and kinetic parameters.

2. Materials and methods

2.1. Organisms, growth conditions and cell- disruption procedure

T. pyriformis, wild strain (from Professor Eduardo Orias, University of California at Santa Barbara, CA) was grown aerobically at 28 8C without shaking to exponential phase in a broth medium containing 1.5%

proteose–peptone, 0.25% yeast extract (Pousada et al., 1979). The exponential phase growth was determined by cell counts.

2.1.1. Metal exposure

The growth of T. pyriformis in the presence of metal salts [cadmium chloride (CdCl

), iron sulphate (FeSO

) and chromium nitrate (Cr (NO

)

9H

O)]

was determined. Erlenmeyer flasks, containing 200 ml of broth medium and heavy metal (cadmium, chromium or iron) at different concentrations, were inoculated with 2 ml of pre-culture of T. pyriformis. A control was carried out under the same conditions but without metal salts. The numeration of cells was done after 72 h of growth.

2.1.2. Preparation of cell-free extracts

After growth, protozoan cells were centrifuged at 4000 g for 10 min. The pellets were washed by Tris buffer (20 mM Tris–HCl (pH 7.5) containing 1 mM PMSF and 1 mM DTT) three times to separate them from the broth medium and were resuspended at a ratio of about 3 ml/g (wet weight) in the same buffer.

Unless otherwise specified, the cells were then disrupted in the cold with a Bandelin Sonopuls Sonifier (90 w, 180 s). The extract obtained was filtered and considered as the crude extract used for all enzyme assays.

2.1.3. Determination of time and number generation

Aliquots of 1 ml were immediately taken (T

) from the control and the exposed cultures, the samples were properly diluted in distilled water and fixed with neutral buffered formalin (NBF) containing 10% (v/v) formalin in phosphate buffer saline (PBS) pH 7 for 1 h.

The cell number was determined by an optical

microscope (Nikon) counting every cell present in

each of six 50 ml sub-samples.

The Tetrahymena populations were characterized by their generation time (g) required to doubling the population. Generation time and generation number were calculated by the following formulas (1) and (2).

Generation time (g):

g ¼ time of growth

number of generations (1)

Generation number (n):

n ¼ ðlog N

log N

Þ

log 2 (2)

where N

is the number of cells at 72 h (time of growth) and N

is the number of cells at T

. 2.1.4. Morphological analysis

Cell morphology was determined under the microscope using a Hitachi CCTV camera fixed in a Leitz Dialux 20 microscope and an acquisition chart (Matrox) established in computer.

2.1.5. Atomic absorption spectrometry

Cadmium, chromium and iron concentrations were determined using an atomic absorption spectrometer (spectr AA-20, Varian model).

2.2. Biochemical assays

All assays were conducted at 25 8C using a Jenway 6405 UV–vis spectrophotometer.

2.2.1. Succinate dehydrogenase

The enzyme was assayed according to King (1967):

100 mM potassium phosphate buffer (pH 7.4), 0.3 mM EDTA, 0.053 mM DCIP and 100 mg of protein. The mixture was pre-incubated 10 min at 25 8C before adding 50 ml of KCN-Succinate (con- taining 3.25 mg/ml of KCN in 0.5 M succinate). The measure of activity was done at 625 nm.

2.2.2. Succinate cytochrome c reductase

Succinate cytochrome c reductase was measured as described by Mackler et al. (1962). The reaction mixture containing 50 mM sodium phosphate buffer (pH 7.4), 2 mM potassium cyanide, 0.625 mg/ml cytochrome c and 100 mg of protein was incubated

during 2 min at 25 8C, then sodium succinate was added at 66 mM as final concentration and the increase in absorbance due to the reduction of cytochrome c was measured. The molar extinction coefficient of reduced cytochrome c is 27,000 cm

M

at 552 nm.

2.2.3. NADPH cytochrome c reductase

NADPH cytochrome c reductase was measured as described by Williams and Kamin (1962). The reaction mixture contained 50 mM sodium phosphate buffer (pH 7.7), 1.25 mg/ml cytochrome c, 0.1 mM EDTA and 100 mg of protein was incubated during 2 min at 25 8C, then 0.1 ml of NADPH (2 mg/ml) was added and the increase in absorbance due to the reduction of cytochrome c was measured at 552 nm (27,000 cm

M

).

2.2.4. Glyceraldehyde 3-phosphate dehydrogenase

GAPDH activity in the oxidative phosphorylation was determined by monitoring NADH generation at 340 nm (Serrano et al., 1991). The reaction mixture of 1 ml contained 50 mM Tricine–NaOH buffer pH 8.5, 10 mM sodium arsenate, 1 mM NAD+ and 2 mM D- G3P. A coupled assay in which aldolase (1 U/ml) produced the stoichiometric breakage of

-fructose 1- 6 biphosphate (2 mM) to

-G3P and dihydroxyacetone phosphate, the first product was the actual substrate of the oxidative reaction.

2.2.5. Acetylcholinesterase

Assay procedure was that of Ellman et al. (1961).

The assay mixture contained 80 mM DTNB, 45 mM acetylthiocholine and 100 mM potassium phosphate buffer (pH 7.4). The absorbance at 412 nm was monitored.

2.2.6. Catalase

The consumption of 7.5 mM H

O

in 50 mM potassium phosphate buffer (pH 7) was monitored at 240 nm (Aebi, 1984).

2.2.7. Glutathione reductase

The assay of Di Ilio et al. (1983) was used. Assay

mixture contained 0.5 mM oxidized glutathione,

1 mM EDTA, 0.1 mM NADPH and 50 mM potassium

phosphate buffer (pH 7.4) and NADPH consumption

was monitored at 340 nm.

2.2.8. Superoxide dismutase

The enzyme was assayed according to Paoletti et al. (1986) with assay conditions: 5 mM EDTA, 2.5 mM MnCl

, 0.27 mM NADH, 3.9 mM 2-mer- captoethanol in 50 mM potassium phosphate buffer (pH 7), monitored at 340 nm. The activity started by the addition of NADH to 0.27 mM as final concen- tration.

2.2.9. Thiobarbutiric acid reactive substances Lipid peroxidation was estimated by the formation of thiobarbituric acid reactive substances (TBARS) and quantified in terms of malondialdehyde (MDA) equivalents according to the method described by Samokyszyn and Marnett (1990): 1 ml of samples was added to 1 ml solution (0.375% thiobarbituric acid and 15% trichloracetic acid in 0.25 M hydrochloric acid).

The tubes were heated at 100 8C during 15 min and they were cooled in the ice to stop the reaction. One then carries out a centrifugation with 1000 g during 10 min. The reading of supernatant was made to 535 nm.

2.2.10.

-b-Hydroxybutyrate dehydrogenase

BDH activity was measured as described by Lehninger et al. (1960) by following NADH produc- tion at 340 nm (e = 6.22 10

M

cm

) using 100 mg of protein in a medium containing: 6 mM potassium phosphate pH 8, 0.5 mM EDTA, 1.27% (v/

v) redistilled ethanol, 0.3 mM dithiothreitol, in the presence of 2 mM NAD+ (Sigma) and 2.5 mg rotenone (final addition) to prevent NADH reoxidation by the respiratory chain. The activity started by the addition of

-b-hydroxybutyrate (Sigma) to 10 mM final concentration.

2.2.10.1. Metal tests. BDH activities were measured as described previously in the absence and the presence of different concentrations of metal salts (cadmium chloride, iron sulfate and chromium nitrate).

2.2.10.2. BDH kinetic studies. Initial velocities of the enzymatic reaction (in the absence and the presence of the metal salts at IC

) were performed by varying the concentration of the substrates, BOH (from 2.5 to 10 mM) or NAD+ (from 0.5 to 2 mM).

Values of the Michaelis constants (K

), the dissocia- tion constants (K

) and the maximal velocity for the

oxidation of BOH and the reduction of NAD+ by the BDH were obtained by mathematical analysis according to the method of Cleland (1963).

2.2.10.3. Western-blotting. After SDS-PAGE (12%) (Laemmli, 1970) and subsequent transfer in nitrocel- lulose (Towbin et al., 1992), the mitochondrial proteins (50 mg) were exposed to 1/100 dilution of monospecific polyclonal anti-BDH antibody and were detected with the secondary antibody of anti-rabbit, IgG peroxidase conjugate (diluted to 1/2500) (Pro- mega).

2.2.11. Protein assay

Protein content was measured according to the Bradford procedure by using bovine serum albumin (BSA) as standard (Bradford, 1976).

2.3. Statistical data analysis

The experimental data represent the mean of four independent assays. Student t-test was used; a p-value lower than 0.05 and 0.01 was considered significant.

The calculation of the inhibition concentrations (IC) was carried out by the analysis of probit (Bliss, 1935).

2.4. Chemicals

DL

-b-Hydroxybutyrate (sodium salt) was pur- chased from Fluka (Buchs Switzerland); NAD

(free acid) and NADH were from Boehringer (Mannheim, Germany); succinate and cytiochrome c were from Sigma (St. Louis, USA) and all other chemicals were of analytical grade.

3. Results

3.1. Inhibitory concentrations, generation number and generation time determination

Fig. 1 shows the sensitivity of T. pyriformis to the presence of metal salts and this sensitivity depends on the metal and the concentration used. Indeed, the three metal salts studied activated the growth of T.

pyriformis at lower concentrations but were toxics

at intermediate and higher level. The inhibition

concentrations (IC) were calculated from Fig. 1 by

the analysis of probit (Bliss, 1935), revealing that cadmium chloride is the most toxic (IC

= 0.034 mg/l and IC

= 0.18 mg/l) compared to iron sulphate (IC

= 0.06 mg/l and IC

= 0.21 mg/l) and chro- mium nitrate (IC

= 1.63 mg/l and IC

= 2.12 mg/l).

In our culture conditions, the density of T.

pyriformis at the beginning of the experiment (T

) was 7.5 10

cells/ml and the normal generation time of T. pyriformis after 3 days of growth was about 21.6 h. Addition of metal salts affected Tetrahymena generation number and generation time (Table 1).

The results obtained indicate that iron sulphate decrease the Tetrahymena growth more than cad- mium chloride and chromium nitrate, respectively (Table 1).

3.2. Effects and accumulation of metal salts on T.

pyriformis

The comparison was done on the effects of the exposition of T. pyriformis to cadmium chloride, iron sulphate and chromium nitrate. Fig. 2 shows that iron sulphate and cadmium chloride modify the morphology of T. pyriformis more than exposure to chromium nitrate. Indeed, cadmium chloride has caused the decrease on Tetrahymena cell’s size and the reduction of its inner volume. In presence of iron sulphate, we can see different forms of T. pyriformis (elongated and circular). No changes were observed with chromium nitrate. Their changes can be caused by metals accumulation in the cell as described in Table 2. The results reveal that the metal salts used were fixed by Tetrahymena cells at concentra- tion dependent manner. In addition, the fixation high concentration of metal salts on Tetrahymena

Fig. 1. Effects of cadmium chloride (A), iron sulphate (B) and chromium nitrate (C) on T. pyriformis growth. The number of Tetrahymena cells was determined in the presence of different concentrations of metal salts. Values are given as means of four separate experiments standard deviations. For experimental con- ditions, see Section

Table 1

Determination of the generation number and generation time of Tetrahymena pyriformis exposed to three metal salts (cadmium chloride, iron sulphate and chromium nitrate) at 10 and 50% of Tetrahymena growth inhibition

Generation number Generation time (h)

IC

IC

IC

IC

Control 3.32 0.16 21.6 1.45

Cadmium chloride 3.04 0.17 2.3 0.11

23.6 1.91 31.2 2.8

Iron sulfate 3 0.53 1.95 0.34

24 1.28 36.9 2.63

Chromium nitrate 3.24 0.18 2.61 0.21

22.2 1.85 27.5 2.12

Values are given as means of four separate experiments standard deviations. For experimental conditions, see Section

*

Significantly different from the control value at p

0.05.

**

Significantly different from the control value at p

0.01 (Student t-test).

Fig. 2. Microscopy images of T. pyrifromis control and exposed at 50% of cell growth inhibition to cadmium chloride (0.18 mg/l), iron sulphate

(0.21 mg/l) and chromium nitrate (2.12 mg/l) at two magnifications (20 and

cells was probably the cause of their mortality (Fig. 1).

3.3. In vivo effect of heavy metals on response of antioxidants defenses markers and metabolic enzymes

The exposure of T. pyriformis to metal salts at two concentrations (IC

and IC

) shows that these elements exerted effects depend on the concentration and the salt used (Tables 3 and 4). Table 3 shows that all metal salts induced a significant increase compared to the control on peroxidized lipid level (TBARS or malondialdehyde), catalase, superoxide dismutase (except for iron sulphate at IC

) and glutathione reductase (except for chromium nitrate at IC

and iron sulphate at IC

and IC

). Malondialdehyde is a terminal product of lipid breakdown due to peroxidation

damage and this (and other aldehydes) can be detected by its reaction with thiobarbituric acid.

Table 4 shows that the mitochondrial markers were very sensible to the presence of metal salts. Indeed, the BDH decreases significantly with cadmium chloride but it increases with iron sulphate at IC

and IC

and with chromium nitrate at IC

. For succinate dehydrogenase activity, it is always increasing except for iron sulphate at IC

where the SDH activity decreases significantly. For succinate cytochrome c reductase activity, it is always decreasing except for iron sulphate at IC

.

For microsome marker, NADPH-cytochrome c reductase increase with cadmium chloride at IC

and decrease with iron sulphate. No changes were observed with chromium nitrate.

For cytosol marker, glyceraldehyde-3-phosphate dehydrogenase increase with cadmium chloride at IC

and decrease at IC

. For chromium nitrate, its activity is always increasing. No changes were observed with iron sulphate.

For mobility marker, acetylcolinesterase decrease with cadmium chloride at IC

and increase with iron sulphate at IC

and with chromium nitrate at IC

. 3.4. Effects of metal salts on BDH activity,

expression and kinetic parameters

In order to explain the differences observed concerning BDH activities (Table 3), Western-blotting

Table 2

Determination by atomic absorption of the amount of heavy metals (cadmium, iron and chromium) fixed by Tetrahymena pyriformis cells at 10 and 50% of its growth inhibition

Amount of heavy metals fixed by cell

IC

IC

Cadmium (pg/cell) 3.6 00.29 30.4 04.77

Iron (ng/cell) 55 08 504 063

Chromium (pg/cell) 8.4 00.69 53.5 06.18 Values are given as means of four separate experiments standard deviations. For experimental conditions, see Section

Table 3

Effects in vivo of cadmium chloride, iron sulphate and chromium nitrate at 10 and 50% of Tetrahymena growth inhibition on response of antioxidant biomarkers

Control Cadmium chloride Iron sulphate Chromium nitrate

IC

IC

IC

IC

IC

IC

Thiobarbutiric acid reactive substances (nmol/mg of protein)

1.07 0.14 4.62 0.37

5.78 0.34

2.1 0.09

2.95 0.21

2.45 0.17

2.56 0.23

Catalase (mmol/min/mg of protein)

0.289 0.30 2.33 0.82

1.99 0.52

1.28 0.57

1.98 0.49

1.44 0.55

0.702 0.37

Superoxide dismutase (mmol/min/mg of protein)

0.18 0.04 0.74 0.08

2.02 0.17

0.1 0.02 0.41 0.06

0.35 0.04

0.57 0.04

Gluthatione reductase (nmol/min/mg of protein)

0.45 0.06 1.55 0.11

1.06 0.09

0.35 0.04 0.48 0.03 0.55 0.04 0.73 0.05

Values are given as means of four separated experiments standard deviations. For experimental conditions, see Section

*

Significantly different from the control value at p

0.01 (Student t-test).

was carried out (Fig. 3). Its analysis revealed the decrease of the BDH expression with cadmium chloride and a light increase of the BDH expression with iron and chromium exposure.

The effects in vitro of cadmium chloride, iron sulphate and chromium nitrate on the BDH activity were also done (Fig. 4). The results obtained showed that all metal salts decrease the BDH activity. The determination of inhibition concentration at 50% by probit analysis (Bliss, 1935) indicates that among the three metals studied cadmium is the most toxic (0.005 mg/l) compared to iron (0.48 mg/l) and chromium (0.52 mg/l).

BDH kinetic parameters: V

, K

BOH, K

NAD+

and K

NAD+ are reported in Table 5. Interestingly, they showed that the kinetic constants with respect to the NAD+ and BOH were modified in the exposed Tetrahymena extract compared to the control. Indeed, the V

of BDH decreases significantly with all metal salts. Also, with cadmium treatment, only K

NAD+ was increased (1.64); with chromium treatment, both K

NAD+ and K

BOH was increased (3.75 and 3-fold, respectively) while K

NAD+

was decreased (0.7); with iron treatment, K

NAD+ increases (2.53) and K

NAD+ decreases (0.41).

4. Discussion

Many aquatic organisms thrive and reproduce in polluted waters. This fact indicates that they are well equipped with defense systems against many toxic xenobiotics simultaneously present in the environ- ment.

Human activities strongly increase the background levels of toxic trace metals in natural waters. Chemical analyses allow the determination of the degree and the nature of pollution, but they do not provide evidence as to the biological consequences (Chapman et al., 1987). Bioassays allow the detection of these effects by measuring the biological responses.

In this work we used T. pyriformis, the most widely used protozoan as a model cell system in morphogen- esis, conjugation, gene mapping, cell division and

Fig. 3. Western blots of the BDH from T. pyriformis at 0, 10 and 50% of T. pyriformis growth inhibition. SDS-PAGE was assayed with 50

Fig. 4. Effects in vitro of three metal salts [cadmium chloride (A),

iron sulphate (B) and chromium nitrate (C)] on the BDH activity of

T. pyrifromis. Values are given as means of four separate experi-

ments standard deviations. For experimental conditions, see Sec-

tion

growth division (Wheatley et al., 1994) to study the impact of three metal salts (cadmium chloride, iron sulphate and chromium nitrate) on metabolism and stress antioxidant systems. Cadmium is a well-studied environmental toxicant. The neurotoxicity of cad- mium is dangerous especially in the developing human brain, because it is thought that cadmium exerts long-term and irreversible effects. Iron is essential for many cellular processes, yet it also has a strong damaging potential. The major part of the cellular iron is safely bound in ferritin as well as in haem- or iron sulphur-cluster containing proteins (enzymes). Chromium is an essential nutrient required

for normal glucose and lipid metabolism and insufficient dietary chromium has been associated with type 2 diabetes and cardiovascular diseases (Anderson, 1989). However, at high concentrations, chromium induces an oxidative stress that results in oxidative deterioration of biological macromolecules (Stohs et al., 2000).

The impact of cadmium chloride, iron sulphate and chromium nitrate on the Tetrahymena growth was determined (Fig. 1). Growth has been widely used as an indicator of pollution stress in marine invertebrates, e.g. bivalves (Widdows et al., 1982; Page and Widdows, 1991) and gastropods (Wo et al., 1999)

Table 4

Effects in vivo of cadmium chloride, iron sulphate and chromium nitrate at 10 and 50% of Tetrahymena growth inhibition on response of metabolic enzymes and subcellular markers

Control Cadmium chloride Iron sulphate Chromium nitrate

IC

IC

IC

IC

IC

IC

b-Hydroxybutyrate

dehydrogenase

(nmol/min/mg of protein)

1.65 0.13 0.71 0.08

0.05 0.09

2.34 0.21

2.99 0.18

3.65 0.33

1.4 0.22

Succinate dehydrogenase (mmol/min/mg of protein)

54 5.8 112 12

136 11

90 6.8

24 3.1

118 7.9

156 6.8

Succinate cytochrome c reductase (mmol/min/mg of protein)

2.77 0.19 0.24 0.04

0.50 0.04

2.11 0.33 1.53 0.15

0.76 0.09

0.48 0.06

NADPH cytochrome c reductase (nmol/min/mg of protein)

1.22 0.09 1.60 0.13

1.11 0.07 0.54 0.07

0.78 0.06

1.21 0.08 1.07 0.11

Glyceraldehyde 3-phosphate dehydrogenase

(mmol/min/mg of protein)

0.27 0.04 0.86 0.06

0.13 0.03

0.35 0.07 0.25 0.05 1.3 0.09

0.77 0.06

Acetylcholinesterase (nmol/min/mg of protein)

0.11 0.02 0.06 0.03 0.05 0.01

0.16 0.04 0.18 0.03

0.26 0.05

0.13 0.02

Values are given as means of four separated experiments standard deviations. For experimental conditions, see Section

**

Significantly different from the control value at p

0.01.

*

Significantly different from the control value at p

0.05 (Student t-test).

Table 5

Determination of the kinetic parameters of Tetrahymena pyriformis BDH in absence and presence of metal salts at 50% in vitro BDH inhibition [cadmium chloride (4.5 ng/l), iron sulphate (0.58

V

(nmol/min/mg of protein) K

NAD+ (mM) K

NAD+ (mM) K

BOH (mM)

Control 2.42 0.07 0.28 0.03 0.53 0.02 0.6 0.05

Cadmium chloride 1.02 0.03

0.22 0.06 0.87 0.18

0.52 0.04

Iron sulphate 0.94 0.03

0.71 0.08

0.22 0.05

0.7 0.06

Chromium nitrate 1.3 0.05

1.05 0.07

0.37 0.09

1.82 0.11

Experiments were varying NAD+ concentration (0.5, 1, 1.5 and 2 mM) or BOH concentration (2.5, 5, 7.5 and 10 mM). Values are given as means standard deviations of four independent experiments.

*

Significantly different from the control value at p

0.01 (Student t-test).

providing a measure of environmental quality. The results show that T. pyriformis is an excellent resistant ciliate compared to Daphnia magna for cadmium chloride and chromium nitrate exposure. Indeed, the IC

of cadmium chloride for T. pyriformis is 180 mg/l compared to 0.08 mg/l for D. magna. For iron sulphate, the IC

for T. pyriformis is 0.21 mg/l compared to 5.9 mg/l for D. magna. For chromium nitrate, the IC

for T. pyriformis is 2.12 mg/l compared to 1.2 mg/l for D. magna.

Also, Fig. 1 reveals that cadmium is more toxic than iron and chromium and that all of these metal salts induce an inhibition effect on cellular divisions.

This inhibition provokes an important increase in generation time and a significant decrease in genera- tion number (Table 1).

Environmental research that started in the 60s has revealed that many living organisms can accumulate certain toxicants to body concentrations much higher than present in their environment. This makes chemical analyses in body tissues much more amenable and affordable. The idea that the measurement of body concentrations in such organisms could be used in routine monitoring procedures was thus established. Nowadays, many monitoring programs around the world use so-called

‘‘sentinel species’’ such as mussels and oysters as indicators of contaminants in marine and estuarine environments. This practice continues in many parts of the world today. Body contaminant concentra- tions can further be used to assess the uptake of contaminants by living organisms and the increase in concentration of a pollutant from the environment to the organism (i.e. bioaccumulation). The bioac- cumulation process may cause significant enrich- ment of contaminant concentrations in body tissues.

Furthermore, once sequestered into the food chain the contaminants may adversely affect species at higher trophic levels.

So, the inhibition of Tetrahymena divisions by metal salts was proved by atomic absorption spectro- metry (Table 2). The results indicate that the amount of metal salts fixed by Tetrahymena cells was concentration-dependant. At high concentrations of metal salts, the amounts fixed by cells are higher and the cell divisions are lower. This can be explained by the fact that the increase in the concentration of metal salts in the cells perturb the cellular divisions and

certainly their metabolism systems and consequently cause their mortalities.

In order to see the metal salts effects on Tetrahymena forms, some pictures were provided (Fig. 2). They reveal that the metal salts induce an important cell structural change: the cadmium chloride causes the decrease on Tetrahymena size and reduction of its inner volume. In the presence of iron sulphate, we can see different forms of Tetrahymena (elongated and circular). No cell structural changes were observed with chromium nitrate. We also observe that cadmium chloride significantly decreasing the mobility of Tetrahymena (data not shown) compared to iron sulphate and chromium nitrate. So, the changes in Tetrahymena form and mobility can play probably an important role in environmental monitoring and protection against metal pollution.

Secondly, it is suggested that biomarkers can be used to devise rapid, effective screening assays, which can complement other testing techniques by significantly reducing the number of samples that may require a more elaborate, definitive or specific evaluation. In this context, biomarker-based techniques do have a major role to play in the overall effort of environmental monitoring and protection. In recent years, there has been a rapid development of enzymatic biomarkers.

This is due not only to advances in biochemistry but also to modern methods of measurement.

In this work, the effects of three metal salts on

Tetrahymena stress biomarkers and metabolic sys-

tems were determined (Tables 3 and 4). The presence

of metal salts on Tetrahymena cells induces a

significative increase in stress markers (superoxide

dismutase, catalase, glutathion reductase and TBARS

or malondialdehyde) (Table 3). The increase in

activities of antioxidant defenses indicates that the

generation of oxygen free radicals by metal salts

should profoundly increase (Maria et al., 1998). The

glutathione reductase eliminates the oxygen free

radicals by the generation of reduced glutathione

(GSH). Also, the superoxide dismutase transforms the

oxygen free radicals on H

O

which is converted by

catalase on H

O. These three enzymes (and others)

play an important role in protection of lipids, proteins

and DNA to destruction and cells to mortality. At

higher concentrations of metal salts, when the

damages are higher and the antioxidant system

cannot protect the cell components, the cell die (Moore, 1985).

The results obtained in Table 3 revealed that cadmium chloride, iron sulphate and chromium nitrate exerted different effects which depend on the concentration on metabolic enzymes (succinate cytochrome c reductase,

-b-hydroxybutyrate dehy- drogenase, succinate dehydrogenase, NADPH-cyto- chrome c reductase and glyceraldehydes-3-phosphate dehydrogenase) and mobility marker (acetylcholines- terase) considered by several authors as being a specific indicator of exposition to the pesticides (Bocquene et al., 1990).

The differences observed in their activities with respect to metal concentrations were due to their properties of permealization and their affinities with respect to various proteins and consequently differ- ent organelles. Indeed, for many metals, the accumulation in the cytosol fraction of the cell indicates a link between functional macromolecules and metal, and can be correlated to the toxic effects.

For example, the cadmium aptitude to induce the metallothionein synthesis, accumulation in the cytosolic protein fractions could explain the high concentration levels measured in cytosol. So, the microlocalisation of pollutants in main organelles are required to understand the mechanism by which aquatic animals respond to pollutant exposure (Simon et al., 2005).

In order to study profoundly the effects of the three metal salts on mitochondria, we choose as model the

D

-b-hydroxybutyrate dehydrogenase (BDH): inner membrane bound enzyme. Table 4 indicated that in vivo, cadmium chloride exerted an inhibition effect on BDH. This effect was due to inhibition of BDH expression (Fig. 3). This inhibition can also be caused by the variation on affinity of BDH towards NAD+

and BOH (Table 5). Also, Table 4 indicated that iron and chromium (at IC

) increased BDH activity. This increase was due to a light increase in BDH expression (Fig. 3). Also, we can say that this increase was especially due to the activation of mitochondrial biogenesis in order to produce NADH and NADPH for cell detoxification. These coenzymes are used by many enzymes as glutathione reductase to produce reduced glutathione in order to eliminate the reactive oxygen species (ROS) toxics for cells produced by metal salts.

5. Conclusion

Biomarkers are thus the ideal tool for biomonitor- ing programmes for pollutants, particularly persistent organic contaminants, in developing countries with the caveat that they must be used correctly. Herein, we provide the framework including considerations and limitations that will allow an environmental manager to design and implement a biomarker-based monitor- ing programme.

In toxicological studies, a single bioassay cannot provide the full picture of the effects in an organism.

Morphological, physiological and biochemical assays were used to provide global information for metal salts cytotoxicity. The results obtained in this work revealed that the metal salts tested caused perturbations in the physiological state of T.

pyriformis. The effects are manifested by changes in response of all antioxidants biomarkers and metabolic enzymes. The results in vivo demonstrated a significant effect of metal salts at all organelles of T.

pyriformis (mitochondria, microsome and cytosol).

This result is very important because the structure unicellular of this ciliate can simulate the effect of pollutants at the eukaryotic cell. Then, we can propose T. pyriformis like cellular model for the study of the toxic impact of different substances in laboratory.

Acknowledgments

This work was supported by the Regional Council of Burgundy and IFR no. 100 and EU grant PL 512 018, and by the ‘‘Programme The´matique d’Appui a` la Recherche Scientifique-Morocco, Biologie no.134’’, and by the ‘‘Action inte´gre´e franco-marocaine MA/05/

134’’.

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