1. P-GLYCOPROTEIN MEDIATED EFFLUX OF CONTAMINANTS IN EARTHWORMS EXPOSED TO DAIRY SHED EFFLUENT
Jeff Brown1, Giuliana Bernardi2, Ravi Gooneratne2, Nicklas Paxeus3, Lars Förlin4 & D.G. Joakim Larsson1.
1Dept. Neuroscience & Physiology, the Sahlgrenska Academy, University of Gothenburg, Sweden, 2Lincoln University, New Zealand, 3Gryaab, Gothenburg, 4Dept. Zoology, University of Gothenburg, Sweden.
INTRODUCTION
P-Glycoprotein (P-gp) are cell membrane transporter proteins enabling organisms to excrete a range of contaminants. However, these efflux transporters are quite ‘non-specific’ in terms of recognised substrates. Blockage of P-Gp by high affinity ‘inhibitors’ may lower excretion and increase toxicity of other pollutants i.e. chemosensitisation, or P-Gp may simply be overwhelmed by a multitude of lower affinity substrates during complex environmental exposures e.g. to treated effluents or sewage sludge. P-Gp are widely evolutionarily conserved and have been identified in annelid worms1. We theorise that P-Gp in earthworms are affected when exposed to chemosensitising agents such as veterinary or human pharmaceuticals contained in treated effluents or sludges are disposed of via pasture application.
1H NMR-metabolomics allows changes in the profile of small metabolites (the ‘metabolome’) to be investigated. It has previously been applied to earthworms in ecotoxicological contexts2,3 and may be a suitable complimentary technique to investigate changes in P-Gp activity.
AIM:
Investigate using 1H NMR-metabolomics the effects of exposure to dairy shed effluent on the earthworm metabolome and links to P-Gp activity.
CONCLUSIONS:
Earthworm tissue extracts showed significant differences in 1H NMR-metabolomic profiles for dairy shed effluent exposed worms, confirming the techniques usefulness.
The altered metabolome is consistent with the hypothesis that the effluent exposed worms were experiencing environmental stress.
P-gp inhibition occurred in effluent exposed worms.
METHODS:
Field sampling of earthworms exposed to dairy shed effluent irrigation (Fig. 1)
P-Gp activity measured by Rhodamine B dye accumulation and 1H NMR-metabolomics
2a) 1H NMR spectra
Aqueous
Lipid
RESULTS:
NMR spectra were dominated by HEFS3, amino acids and long-chain fatty acids (Fig. 2a).
Significant differences between dairy effluent exposed and control earthworms could be seen in both aqueous and lipid metabolites (Fig. 2b).
Differences in dye uptake suggested P-gp may be involved.
H2O
alanine
leucine
betaine
fumarate
maltose
* HEFS
(2-hexyl -5-ethyl-furansulfonate) *
acetylcholine (?)
Solvent Peaks
lipid CH2
lipid CH3
olefinic methines
unknown
2b) PLS-DA analysis
Aqueous
Lipid
Dairy effluent exposed
Organic farm control
R2X=0.70
R2Y=0.89
Q2=0.65
More alanine, glutamine, glutamate, betaine & fumarate
More HEFS, leucine & maltose
Dairy effluent exposed
Organic farm control
More fatty acids
R2X=0.78
R2Y=0.96
Q2=0.69
Less fatty acids
** (p<0.01)
Fig. 1. Field sampling of worms exposed to irrigated dairy shed effluent in New Zealand.
Inset top - Aporrectodea caliginosa.
Inset bottom – exposing worms to rhodamine dye for P-gp activity assay
Fig. 2 a) 1H NMR spectra of aqueous and lipid extracts from earthworms exposed to dairy shed effluent. Dominant metabolites are indicated including the earthworm specific 2-hexyl-5-ethyl-furansulfonate (HEFS).
Fig. 2 b) Partial Least Squares-Discriminant Analysis (PLS-DA) showing good class separation between dairy effluent exposed and control earthworms. Major metabolites responsible for the class separations are shown.
Fig. 3. Rhodamine uptake assay showing greater dye accumulation (less P-gp activity) in dairy shed effluent exposed worms. Addition of P-Gp inhibitor Cyclosporin A, fully inhibits P-gp removing the difference.
REFERENCES 1 Smith JM & Prichard RK (2002) Localisation of P-glycoprotein mRNA in the tissues of Haemonchus contortus adult worms and its relative abundance in drug-selected and susceptible strains. J. Parasitology 88 (3): Jones OAH et al., (2008) A metabolomics based approach to assessing the toxicity of the polyaromatic hydrocarbon pyrene to the earthworm Lumbricus rubellus. Chemosphere 71: Bundy JG et al. (2002) Metabonomic assessment of toxicity of 4-fluoroaniline, 3,5-difluoroaniline and 2-fluoro-4-methylaniline to the earthworm Eisenia veneta. Environ. Toxicol. Chem. 21(9):