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Exocellular oxidases can be sensitive and selective biomarkers of fungal exposure to metals
Jérémie Lebrun, Isabelle Lamy, Christian Mougin
To cite this version:
Jérémie Lebrun, Isabelle Lamy, Christian Mougin. Exocellular oxidases can be sensitive and selective
biomarkers of fungal exposure to metals. 8. EMEC8 European Meeting on Environmental Chemistry,
Dec 2007, Inverness, United Kingdom. 1 p, 2007. �hal-02815418�
Exocellular oxidases can be sensitive and Exocellular oxidases can be sensitive and selective biomarkers of fungal exposure to metals selective biomarkers of fungal exposure to metals
J Jé ér ré émie Lebrun, Nelly Wolff, Isabelle Lamy mie Lebrun, Nelly Wolff, Isabelle Lamy and and Christian Mougin Christian Mougin
Unit Unité é de Physico- de Physico -chimie et chimie et Ecotoxicologie Ecotoxicologie des des SolS SolS d d’ ’Agrosyst Agrosystè èmes mes Contaminé Contamin és, s, INRA de Versailles, France
INRA de Versailles, France INTRODUCTION INTRODUCTION
Soil pollution by heavy metals is a worldwide problem. Only a few tests are available to assess the effect of pollutant on soil microorganisms, and more widely on this ecosystem. Metals can modulate the expression of enzymes secreted by microorganisms and involved in soil functioning.
Methods based on enzymatic activity measurements may offer promising perspectives for ecotoxicological risk assessment in soils. Because of their ecological pertinence, we retain filamentous fungi for the development of enzymatic methods.
OBJECTIVES OBJECTIVES
METHODS METHODS
- - laccaselaccase --lignin peroxidaselignin peroxidase
-
-Mn-Mn-peroxidaseperoxidase
RESULTS RESULTS
CONCLUSION CONCLUSION
Trametes versicolor responds to heavy metals by morphological modifications and modulations of enzymatic expression. Cadmium, copper and mercury are able to induce the production of exocellular oxidases. Since the enzymatic responses carry out at environmental levels of metals, these
enzymes can be biomarkers of fungal exposure to metals in contaminated soils. Further studies are now necessary to improve enzyme extraction from soils.
I
I-- METAL TOXICITY AND MORPHOLOGICAL RESPONSESMETAL TOXICITY AND MORPHOLOGICAL RESPONSES
- Cultures of Trametes versicolor in basic liquid medium during two weeks - Addition of Cu, Zn, Cd, Pb or Hg solutions during the cultivation (3thday)
- Determination of dry biomass used as toxicity/tolerance indicator at the end of cultivation - Observations in scanning electron microscopy to characterize the morphological responses II
II-- RESPONSES OF EXOCELLULAR OXIDASESRESPONSES OF EXOCELLULAR OXIDASES
- Each metal is added in basic medium at three concentrations (Cutot= 0.01, 0.1 and 1 mM) - Measurements of oxidase activities in culture media during the cultivation
(after three days, one or two weeks exposure) III
III--SENSITIVITY OF ENZYMATIC RESPONSESSENSITIVITY OF ENZYMATIC RESPONSES
- Use of a low complexing liquid medium (suppression/diminution of some nutritive elements known as metal ligands) to raise the bioavaibility
- Addition of Cu (from 1 nM to 1 mM) or mixtures of metals (0.1 mM for each metal) - Measurements of activities in culture media at defined times
I-I-METAL TOXICITY AND MORPHOLOGICAL RESPONSESMETAL TOXICITY AND MORPHOLOGICAL RESPONSES II-II-RESPONSES OF EXOCELLULAR OXIDASES (exposure for two weeks)RESPONSES OF EXOCELLULAR OXIDASES (exposure for two weeks) The present study aims at assessing both selectivity and sensiti
The present study aims at assessing both selectivity and sensitivity of the response of exocellular oxidases secreted by the vity of the response of exocellular oxidases secreted by the basidiomycete
basidiomycete Trametes versicolorTrametes versicolorexposed to metals.exposed to metals.
For that purpose, the activities are monitored in the presence of metals and in media exhibiting different levels of metal complexation. Exposures with mixtures of metals are designed to reflect soil multiple contaminations. Threshold metal toxicity to this fungus were determined.
IIIIII--SENSITIVITY OF ENZYMATIC RESPONSES (exposure for two weeks)SENSITIVITY OF ENZYMATIC RESPONSES (exposure for two weeks)
In spite of morphological impacts, In spite of morphological impacts, T. versicolorT. versicoloris is tolerant of heavy metals
tolerant of heavy metals
- Laccase and Mn-peroxidase activities are increased by copper and cadmium at the total dose of 1 mM (Fig. 2a and 2b).
- The response to mercury is more sensitive with an increase in activities from 0.01 mM (At 1 mM, the fungus doesn’t grow).
- Zinc and lead are without effects on these activities.
- Lignin peroxidase is specifically expressed in the presence of copper.
Expression of exocellular oxidases is modulated by heavy metalExpression of exocellular oxidases is modulated by heavy metalss - Metals have no significant effect on the biomass of T.
versicolor except mercury at the total concentration of 1 mM.
- However, metals cause some morphological alterations of mycelia and the synthesis of extracellular matrix (Fig. 1).
Fig. 2a. The values means ± SE (n= 9) ; significant differences *p< 0.05
RESPONSE OF EXO
RESPONSE OF EXO--OXIDASESOXIDASES
CuCu HgHg Pb Pb Zn Zn
CdCd METALLIC EXPOSURE
METALLIC EXPOSURE
Fig. 1. Scanning electron microscopy (magnification, x 5000) of T. versicolor mycelia exposed for two weeks to Cu or Cd Control Cu (1 mM) Cd (1 mM)
Fig. 2b. The values means ± SE (n= 9) ; significant differences *p< 0.05
*
*
*
*
0 2 4 6 8 10
Cu Zn Cd Pb Hg
laccase (U/ml)
Ctrl: without metal 0,01mM 0,1mM 1mM
*
*
*
0,0 0,2 0,4 0,6 0,8
Cu Zn Cd Pb Hg
Mn-peroxidase (U/ml)
Ctrl: without metal 0,01mM 0,1mM 1mM
0 4 8 12 16 20
Basic Low complexing
Media
laccase (U/ml)
Control Cu added at 1 mM
0 5 10 15 20 25 30
1
laccase (U/ml)
control,and each metal added at 0.1 mMCuCu+ZnCu+CdCu+PbCu+Hg
- In the low complexing medium, the presence of free copper species is favoured (determination of Cu2+).
- The production of oxidases increases with the free copper content (Fig. 3).
- In the low complexing medium, the oxidase activities are significantly increased by copper from 1 µM (Fig. 4a and 4b).
- This response of exo-oxidases is more sensitive than in the basic medium where the threshold is 1 mM (Fig. 4 vs Fig. 2).
Fig. 3. The values means ± SE (n= 9).
- Mixtures of metals provided contrasted results.
- In all cases, the oxidase activities increase compared to control (Fig. 5).
The sensitivity and the intensity of enzymatic responses to cThe sensitivity and the intensity of enzymatic responses to copper are increased when metal is more bioavailableopper are increased when metal is more bioavailable
Fig. 5. The values means ± SE (n= 9).
1-1-Comparison of media Comparison of media 22--DoseDose--responses to copper responses to copper 33--Mixtures of metalsMixtures of metals
Cu2+ << 0.001 % Cutot Cu2+ #0.3 % Cutot
EMEC8, Inverness, Dec. 2007 EMEC8, Inverness, Dec. 2007
*
*
* *
0 2 4 6 8 10 12 14 16
Ctrl 1 nM 0.01 µM0.1 µM 1 µM 0.01 mM0.1mM 1mM concentration of copper
laccase (U/ml)
*
*
*
*
0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4
Ctrl 1 nM 0.01 µM 0.1 µM 1 µM 0.01 mM0.1mM 1mM
concentration of copper
Mn-peroxidase (U/ml)
Fig. 4a. and 4b. The values means ± SE (n= 9) ; significant differences *p< 0.05
10 µm 10 µm 10 µm
