Combined effects of temperature and cadmium on membrane phospholipid composition, aerobic and antioxidant capacities and lipid peroxidation in two freshwater fishes.

0 1 2 3 4 5 6 7 8 15°C 15°C -Cd 30°C 30°C-Cd

Combined effects of temperature and cadmium on membrane phospholipid composition,

oxidative and antioxidant capacities and lipid peroxidation in two freshwater fishes

Mariem Fadhlaoui and Patrice Couture

Institut National de la Recherche Scientifique, Centre Eau Terre Environnement, 490 de la Couronne, QC G1K 9A9, Canada

mariem.fadhlaoui@ete.inrs.ca

Introduction

Poikilotherms adapt the composition of their cell membrane phospholipid fatty acids according to temperature (modification of fatty acid unsaturation) to maintain membrane fluidity and function: homeoviscous adaptation (HVA)

Metals can induce reactive oxygen species production and as a consequence, enhanced lipid peroxidation

Membranes enriched with PUFA, typical of cold acclimation, are more prone to lipid peroxidation

Objectives

Evaluate enzymatic indicators of oxidative capacities and antioxidant defense Cd (6μg/l) Without metal Cd (6μg/l) Without metal Cd (6μg/l) Without metal Cd (6μg/l) Without metal Experimental design 9°C 28°C Perca flavescens Pimephales promelas

Methods

Lipid extraction and FAME analysis Chloroform/methanol extraction

Polar lipids Apolar lipids

Esterification (BF₃, 75°C) FAME GC-FID 15°C 30°C Purification

Membrane fatty acid composition

0 10 20 30 40 50 60 70 80 15 15_Cd 30 30_Cd AGPI-Mené AGPI-Perchaude

Cold Cold-Cd Warm Warm-Cd

PUFA-Fathead minnow PUFA-Yellow perch B B A _a a b b Pe rc en ta ge (% )

Cell membrane phospholipids of cold-acclimated fish were enriched in PUFA

Agrees with homeoviscous adaptation to maintain membrane fluidity

Fathead minnow: PUFA increased at 28°C

Yellow perch: PUFA decreased at 9°C and increased at 30°C

Cd exposure modified the normal response

to thermal stress In warm-acclimated fish, PUFA decreased Cd exposure

Oxidative capacity

0 0,01 0,02 0,03 0,04 0,05 0,06 0,07 0,08 0,09 15°C 15°C-Cd 30°C 30°C-Cd CCO CS 0 0,01 0,02 0,03 0,04 0,05 0,06 0,07 0,08 9°C 9°C-Cd 28°C 28°C-Cd CCO CS 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0.09 0.08 0.06 0.04 0.02 A _A B B a _{a a} b a a b b A B AB AB

Fathead minnow Perca flavescens

Cold acclimation: oxidative capacities CS increase in Cd-exposed warm-acclimated

fathead minnows: Cd exposure modified the normal response to thermal stress

In cold-acclimated yellow perch, CCO activity was sharply decreased by Cd

exposure 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9

Cold Cold-Cd Warm Warm-Cd

yellow perch

fathead minnow

Lipid peroxidation (LPO)

_{Fathead minnow}

Yellow perch

Cold Cold-Cd Warm Warm-Cd

a A _A B B ab ab b 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.9 M DA nm ol /m g p ro te in s 0

Cold acclimated fish: PUFA (More prone to lipid peroxidation) MDA increase

Yellow perch

In warm acclimated fish under Cd exposure: despite the high PUFA percentage, MDA was

lower than in cold acclimated fish antioxidant defense efficiency?

Fathead minnow

Antioxidant capacity

0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 15°C 15°C-Cd 30°C 30°C-Cd Fathead minnow Yellow perch

UI SOD CAT GPx MDA CCO CS UI 1 SOD 1 CAT 1 GPx 1 MDA 1 CCO 1 CS 1

UI SOD CAT GPx MDA CCO CS UI 1 SOD 1 CAT 1 GPx 1 MDA 1 CCO 1 CS 1

Membrane lipid remodeling, lipid peroxidation,

oxidative and antioxidant capacity

8 7 6 5 4 3 2 1 0 U I/ m g p ro te in s 4.5 4 3 2.5 2 1.5 1 0.5 0 3.5 U I/ m g p ro te in s Fathead minnow Yellow perch

Cold Cold-Cd Warm Warm-Cd Cold Cold-Cd Warm Warm-Cd

B B A B b a ab b

The activities of antioxidant enzymes were different under the same Cd concentration and thermal stress Interspecific response

CCO activity was positively correlated with the unsaturation index

CCO activity correlated with 18:0 (negative)

20:5n3 (positive) and 22:6n3 (positive)

18:0, 20:5n3 and 22:6n3 are important components of cardiolipin

Our study suggest that changes of fatty acids in the vicinity of CCO may

contribute to the increase of its activity in cold-acclimated fish

P<0.05 P<0.01 P<0.001

Conclusions

Combined exposure to Cd and elevated temperature modified the normal response to thermal stress

Oxidative capacities are affected by changes in temperature and Cd exposure CCO activity is affected by membrane composition

The rate of LPO generally increased with unsaturation

In warm-acclimated perch exposed to Cd, the risk of LPO was likely countered by the action of antioxidant enzymes

U I/ m g pr ot ein s U I/ m g pr ot ein s - - + - - + + + - - + - + - - + - +

*Different superscript letters of the same case (black for fathead minnow; red for yellow perch)

indicate significant differences (Tukey and Wilcoxon (p≤0.05)

SOD GPx

SOD: superoxide dismutase, CAT: catalase, GPx: Glutathione peroxidase, MDA: malondialdehyde, CCO: cytochrome c oxidase,

CS: citrate synthase,

We do not know whether changes in membrane composition that occur during thermal acclimation modify their vulnerability to metal-induced lipid peroxidation

In two species of fish acclimated to different temperatures and exposed or not to cadmium:

Compare phospholipid membrane composition and susceptibility to lipid peroxidation

Examine the relationships between membrane composition, oxidative and antioxidant capacities

A

PUFA: Polyunsaturated fatty acid SFA: Saturated fatty acid

UI: unsaturation index (number of double bonds

in each fatty acid X the mol percentage of that fatty acid)

PUFA SFA

*Different superscript letters of the same case indicate significant differences (Tukey and Wilcoxon (p≤0.05)

CCO activity was also correlated with the unsaturation index

AB B AB B

b

b

a

a

Our study suggests that Cd-induced

modifications of membrane composition and affects CCO activity in

cold-acclimated yellow perch