Download SUPPLEMENTARY MATERIAL Extracts deriving from olive mill

SUPPLEMENTARY MATERIAL
Extracts deriving from olive mill waste water and their effects on the liver of the
goldfish Carassius auratus fed with hyperocholesterolemic diet
Alessio Alescia, Nicola Ciceroa,*, Andrea Salvoa, Deborah Palombierib, Daniele Zacconea, Giacomo
Dugoa, Maurizio Brunoc, Rossella Vadalàa, Eugenia Rita Laurianoa, and Simona Pergolizzia
a
Department of Environmental Sciences, Territorial, Food and Health Security (S.A.S.T.A.S.), Viale
F. Stagno d’Alcontres 31, University of Messina, 98166 Messina, Italy
b
Department of Biological and Environmental Sciences, Viale Stagno d’Alcontres 31, University of
Messina, 98166 Messina, Italy
c
Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF),
Università di Palermo, Viale delle Scienze, Parco d’Orleans II - 90128 Palermo, Italy
Tissue preparation, histology and immunohistochemistry
15-20 goldfish (Carassius auratus) were used for the present study. The fishes were purchased from
a business and kept in 8-liter aquaria under the experimental conditions of the laboratory for 30
days. They are then divided into four groups: the first group was fed with a cholesterol 10%
enriched diet; the second group received a curative treatment, with a hypercholesterolemic diet and
polyphenols, administered only in the last 15 days of treatment; the third group was fed with
hypercholesterolemic diet with additional administration of polyphenol extracts from Olea
europaea as a preventative treatment; finally, the fourth group normal-fed, was used as control. At
the end of the experiment, the fishes were anesthetized with Ethyl 3-aminobenzoate
methanesulfonate (MS) and we proceeded with the removal of organs of interest: liver. The samples
were processed according to the techniques of sustainable construction preparations for light
microscopy. They were then fixed in immunofix® (Bio-Optica, Milano, Italia) for two hours and
dehydrated in ascending series of alcohols up to immersion in xylene. It was done with subsequent
inclusion in Bioplast® (Bio-Optica, Milano, Italia) and microtome rotary cutting, resulting sections
with a thickness of about 3-5 micrometers. The sections thus obtained were placed on glass slide
and left in the oven for 48 hours. Then a morphological analysis was carried out by performing twotoned histological staining: hematoxylin-eosin, and histochemical staining: Periodic acid Schiff
reaction, May Grunwald-Giemsa and Sirius Red. And even to confirmed this results , the research
has made use of immunohistochemical techniques, testing CYP1A, S100 and α-Smooth with optic
and confocal microscope for observation.
Panel S1. H/E, 100x. a) Normal group; b) Hypercholesterolemic group: the epatocytes are irregular, it is evident
cellular ballooning and lipid droplets, and centrilobular veins are alterated; c) Preventive group: it is evident the
presence of lipocromics pigments; d) Curative group: histology tend to normal; PAS, 100x. e) Normal group; f)
Hypercholesterolemic group; g) Preventive group; h) Curative group.
Panel S2. Sirius Red, 100x. a) Normal group; b) Hypercholesterolemic group: the collagen is more present around
centrilobular veins and in the gaps between hepatocytes, to prove a fibrotic condition; c) Preventive group; d) Curative
group: in this group it s possible to observe a decreasing concentration of collagen to demonstrate positive effects of
polyphenols.
Panel S3. CYP1A, 100X. a) Normal group; b) Hypercholesterolemic group : it is evident the marking around
centilobular veins and in some hepatocytes; c) Preventive group ; d) Curative group: the marking is absent to explain
beneficial effect of polyphenols.
Panel S4. May Grunwald-Giemsa, 100x. a) Normal group; b) Hypercholesterolemic group : there are many
granulocytes to prove immune response to oxidative stress cholesterol-induced. c) Preventive group; d) Curative group:
it is possible to observe a reduction of the number of granulocytes in response to effect of polyphenols. S-100, 100x. e)
Normal group; f) Hypercholesterolemic group: the granulocytes are marking positively because they present a specific
protein, calprotectin, that plays a role in flogosys and in immune response; g) Preventive group; h) Curative group: the
reduction of granulocytes shows a morphological condition that tend to normal.
Panel S5. α – SMOOTH ACTINE. Peroxidase, 100x. a) Normal group; b) Hypercholesterolemic group: alteration of
centrilobular veins demonstrate presence of a inflammatory condition marked with specific antibody of smooth muscle
of endothelium; c) Preventive group; d) Curative group. Fluorescence, 100x. e) Normal group; f) Hypercholesterolemic
group; g) Preventive group; h) Curative group. This results confirm data obtained with peroxidase, explaining and
proving alteration of veins to response to oxidative stress cholesterol-induced at level of smooth muscle of endothelium
in centrilobular veins.