New results on Senifood project sponsorized by Nutrafur S.A..

1. May 24-27, 2011 . Murcia· Spain
I r tian l y n
Trace Elements &Health
may 2011 I murcia spain

2. I
Poster P.5.1.
Potassium apigenin: a promising
radiation countermeasure
(llMiguel Alcaraz; (4.IJulián Castillo; (4J Obdulio Benavente Carda;
(2.IDavid Armero; (l)Miguel Alcaraz-Saura; (5}(,IDaniel Achel;
(3Vicente Vicente and (6)Manuel de las Heras
Departments of (l)Radiology and Physical Medicine, (2)Nursing and (3)PathoJogy,
University of Murcia. 30100-Campus de Espinardo. Murcia.
(4)Department l+D+ i u trafur S.A., Camino Viejo de Pliego s/n
3Ü320-Alcantarilla, Murcia.
(5)Applied Radiation Biology Cen.h'e, Radiological and Medica) Sciences
Research Inslitute, Ghana Atomic Energy Commission, Legon-Acera, Ghana.
(6l[)epartment of Radiotherapy. Clinic Hospital oí San Carlos. University
Complutense oí Madrid. CI Prol. Mart n Lagos sI n, 28040-Mad.rid.
The genoprotective and radioprotective eifects of potassium
apigenin against chromosomaJ damage induced by ionizing
radiation were compared wíth those oí L-ascorbic acid,
o-tocopherol, diosmin, rutin and the S-containing compounds
(dimethyl sulfoxide (DMSO) and Amifostine), using the
micronuc1eus test for antimutagenic activity to evaluate the
reduction in the frequency oí micronuclei (MN) in cytokinesis­
blocked cells of human lymphocytes befare and after irradiation;
and the ceH survival using the ceH viability test with MTT in
201

3. Posters
tvw prostate cell lines (PNT2, TRAMP-Cl) administered before
exposlue to different X ray doses (OGy, 4Gy, 6Gy, SGy and 10Cy).
The results pomt to the significant genoprotective capacity of the
substances assayed before radiabon: Apigenin (AP) -~ Diosmin
(D) = aAocopherol (E) = L-ascorbic acid (C) = Amifostine (AMIF)
(p<O,OOl» Ruhn (R) = Dimetil sulfoxide (DMSO) (p<O,05»
Irradiated Controls (CO, the maximurn. protection factor attained
being 50%. When the substances were administered imrnediately
after radiation, th re ults were different: AP= E (p>O.OO1» 0=
C (p>O.05»Ci, with a maximum protection factor of 38%. In
contrast, AMIF, R and DMSO lost their genoprotective effect.
Cell survival obtained with 20 ,uM and 40 ~{M potassium
apigenin adminjstered before radiatian with up to 10 Gy showed
a Protection Factor oí 100%, reducing radioinduced cell death by
30.8% in both celllines assayed (p<O.OOI). Apigenin showed the
greatest pre-treatment pratective effect a good post-treatment
protection capacit}o and no toxicity towards censo
For this reason, our results indicate that apigenin may be
developed as a radioprotectant for humans against the potentially
damaging effects of exposure to radiabon. Human clinical trials
examining the effect of supplementation oí potassium apigenin
on disease prevention have not been conducted, although there
is considerable potential far apigenin to be develaped as a
radioprotectíve agent.
Iros report was supported by a grant fram th Nationa1 Spanish
R + D Programme CE IT of the Spanish Ministry of Science and
Tedu1010gy (acronym: SENIFOOD) and by a Fello''ship oí the
lnternational Atomic Energy Agency (GHA 10021).
202

4. POTASSIUM APIGENIN: A PROMISING
RADIATION COUNTERMEASURE
((1)Miguel
Alcaraz; (3)Julián Castillo; (3)Obdulio Benavente-García;(1)Miguel
Alcaraz-Saura; (4)(*)Daniel Achel; (2)Vicente Vicente y (5)Manuel de las Heras
Departments of Radiology and Physical Medicine(1) and (2)Pathology, Faculty of Medicine, University of Murcia. 30100-Campus de
Espinardo. Murcia.
(3)Department I+D+ i Nutrafur, Camino Viejo de Pliego s/n 30320-Alcantarilla, Murcia.
(4)Applied Radiation Biology Centre, Radiological and Medical Sciences Research Institute, Ghana Atomic Energy Commission, Legon-
Accra, Ghana.
(5)Department of Radiotherapy. Clinic Hospital of San Carlos. University Complutense of Madrid. C/ Prof. Martín Lagos s/n, 28040-Madrid.
INTRODUCTION cytokinesis-block micronucleus test
(MN) with cytochalasin-B on
This work is carried out as part of our efforts to find a non toxic irradiated human lymphocytes
substance that can be used to reduce the harmful effects induced by
ionizing radiation. This compound could protect against genetic damage,
mutations and tetarogenic lesions that act through free radical production.
Recently substances with characteristic anti-oxidative properties are
gaining attention as potential radioprotective candidates. In this study we
assay the antioxidant substance potassium apigenin (AP), a habitual
substance in human diet, for its radioprotective capacity against damage
induced by the exposure to X-rays, Its modified structure serves to increase
its solubility water solubility.
MATERIAL AND METHODS Fig. 1: Different types of cells in the microscopic preparations: CB:cytokinesis-
blocked cell; MNCB: cytokinesis-blocked cell with micronúcleos; L: lymphocyte;
erythrocyte hemolized.
Chemicals: Apigenin (API) and Diosmine (D) were supplied by Nutrafur S.A. (Murcia, Spain); 99% L-ascorbic
acid (AA) and 99% δ-tocopherol (E) were obtained from Sigma Co. (Madrid, Spain). Dimethylsulphoxide GENOTOXICITY
(DMSO) and Rutin (R) were obtained from Merck (Darmstadt, Germany); and Amifostine (AMF) were obtained
from Schering-Plough, SA (Ethyol® injectable, Madrid, Spain).
AP was studied by means of two routine assays commonly employed in radiological protection:
Genoprotection assay: We used the cytokinesis-block micronucleus test (MN) with cytochalasin-B on
irradiated human lymphocytes to evaluate the genoprotective effects of AP following the technique described
by IAEA (2001). Samples of blood were cultivated at 37°C for 72 hours in 4.5 ml of HAM F-10 medium (Sigma
Co, Madrid) containing 15% fetal bovine serum, 16µg/ml phyitohemaglutinin (Sigma Co, Madrid), 100 µg/ml
MN/500 CB
penicilin/streptomicin (Sigma Co, Madrid) and glutamine 1 µg/ml; cytochalasina B (6µg/ml, Sigma Co.) was
added 44 hours after the inception of the cultivation. At the end of 72 hours the culture was terminated and the
cells harvested. At harvest the culture solution was centrifuged and a hypotonic solution KCl (75μM) was
added to the collected cells. A fixative solution of methanol:acetic acid (3:1) was added and slides prepared.
The preparations were stained with May-Grünwald and Giemsa stains. Scoring was done by counting the
number of MN per 3000 binucleated cells (CB), using a Zeiss light microscope (Oberkochem, Germany) with a
CYTOTOXICITY
magnification of 400X. The test substances were added to the samples of human blood cultures to obtain a
final concentration of 25 μM, before and after the exposure to X-rays.
Control
Cytoprotection assay: We used the MTT cell survival assay to assess cytoprotective effect using two prostate
cell lines PNT2 (normal epithelial cells) and TRAMP-C1 (tumoral cells).The cells were cultivated normally in the
presence of API (20μM and 40μM) for 24 and 48 hr after the exposure to X-rays. Briefly: the cells were
(p<0,001) versus C Treatments
incubated in 200 μl of medium supplemented with 50 μl of MMT (MTT) (8mg/ml) in 96 well microplates (4h,
37ºC, 5%CO2). After centrifugation (240g for 10 mn) to remove non metabolized MTT, 100 μl of DMSO was (p<0,001) versus Ci
60
added to each well to solubilized the MTT taken up by the live cells. Finally, MTT reduction data were o (p<0,001) versus Ci 55
M Magnitude of Protection (%) )
50
agnitude of protection (%
expressed as absorbance at 570nm and 690nm with a Multiskan MCC/340P spectrophotometer. Control + Apigenin 24h + 48 h
45
An X-ray equipment Andrex SMART 200E machine was used (YXLON International, Hamburg, Germany) 40
operating at 120 kV, 4.5 mA, DFO 36 cm and ambient temperature to administer a dose of 2 Gy for the MN 35
assay and of 0 Gy, 4Gy 6Gy 8Gy and 10 Gy for the cell survival determinations. Statistical analysis consisted 30
of pair wise contrast analysis of variance to compare the percentages of cell survival in the cultures with 25
20
different concentrations of the compounds and contrasting their means with the method of minimum 15
significant differences. The analysis was carried out with a logarithmical transformation of the data to 10
accommodate the adjustments made for the ANOVA. Finally, the results were used to obtain the Magnitude of 5
Protection: Magnitude of protection (%) = ((Fcontrol – Ftreated) / Fcontrol) x 100 (where Fcontrol = frequency of MN in 0
RO CA E AP AA Amif Te R D DMS
irradiated blood lymphocytes and Ftreated = frequency of MN in blood lymphocytes treated pre and post-γ- PF (%) 57.7
PF (%) 57,7 50
50 50
50 50
50 38.5
38,5 38.5
38,5 23,1
23.1 23,1
23.1 23,1
23.1 16,6
16.6
irradiation as described previously) and the Dose Reduction Factor (DRF) determined. Figure 2: Control+Apigenin 24h and 48h
Treatments
Trratamientos
Treatments
RESULTS
Apigenin-K 24h Apigenin-K 24h
Genoprotection assay
The results point to the significant genoprotective capacity of the substances assayed
before radiation: Apigenin (AP) = Diosmin (D) = α-tocopherol (E) = L-ascorbic acid (C) =
Amifostine (AMIF) (p<0,001)> Rutin (R) = Dimethyl sulfoxide (DMSO) (p<0,05)> Irradiated
Controls (Ci); the maximum protection factor attained being 50%. When the substances
were administered immediately after radiation, the results were different: AP= E
(p>0.001)> D= C (p>0.05)>Ci, with a maximum protection factor of 38%; however, in
contrast, AMIF, R and DMSO lost their genoprotective effects.
Cytoprotection assay
In the PNT2 cell line assay AP showed a radioprotective effect by significantly increasing
the cell survival to 10 Gy (p <0.001) after X-ray exposure, this represens a Factor of Doses APIG 20 CONTROL APIG 40 Doses
Protection of 100% and 70% at 24h according to the concentration of AP studied (40 μM
and 20 μM, respectively); these values descended respectively to 61% and 50% at 48h (p Figure 3 : Cellular survival (%) with Apigenin at the 24 hours . (p<0,001 versus Ci).
<0.001), we determined a (DRF) of FRD of between 3.1-3.3 according to the concentration
of AP assayed. Apigenin-K 48h Apigenin-K 48h
In the line TRAMP-C1 assay, AP also showed a radioprotective effect by increasing the
cell survival significantly to 10 Gy after exposure to X-rays (p <0.001), representing a
Factor of Protection of 58% at 24h and increased to 69% and 85% at 48h (p <0.001)
respectively. A Dose Reduction Factor of between 2.5-3 was determined according to the
concentrations of AP assayed.
CONCLUSION
our results indicate that potassium-apigenin may be developed as a
radioprotectant for humans against the potentially damaging effects of exposure
to radiation. Human clinical trials examining the effect of supplementation of Doses
APIG 20 CONTROL APIG 40 Doses
potassium apigenin on disease prevention have not been conducted, although Figure 4: Cellular survival (%) with Apigenin at the 48 hours. (p<0.001 versus Ci).
there is considerable potential for apigenin to be developed as a radioprotective
agent. This report was supported by a grant from the National Spanish R + D Programme CENIT of the Spanish Ministry of Science and
Technology (acronym: SENIFOOD) and by a Fellowship of the International Atomic Energy Agency (GHA10021).

5. Poster P.5.2.
Carnosic a,cid: a promising molecul,a for
radiation protection
11lMiguel Alcaraz; (4 ]ulián Castillo; (4)Obdulio Benavente García;
(2)David Annero; (l)Miguel Alcaraz-Saura; (5)(')Daniel Achel and
(3Vicente Vicente
Departments of (IJRadiology and Physical Medicine, (l)Nursing and (3)
Pathology, University af Murcia. 30100-Campus de Espinarda. Murcia.
(4)Department I+D+ i Nutrafur S.A., Canüno Viejo de Pliego 51 n
30320-Alcantarílla, Murcia.
(5JApplied Radiaban Biology Centre, Radialagical and Medical Sciences
Research lnstitute, Ghana Atamic Energy Comrnission, Legan-Acera, Ghana.
The genoprotectíve effect of carnosic acid against damage índuced
by ionizing radiabon was compared with the effect of several
antioxidant compounds by means of the micronucleus test for
antímutagenic activity, in which the reduchon in the frequency of
micronuclei was evaluated in cytokinesis-blocked cells of human
lymphocytes before and after 2 Gy of 'Y-radiatian. Also, the
radioprotective effect of the most effective compounds was then
studied by a cell viability test (MTT) in the PNT2 (normal prostate)
and B16Flü (m.elanoma) ceH lines when they are administered
befare exposure to X ray doses (OGy,4Gy,6Gy,8Gy and lOGy).
The results point to the significant genoprotective capacity
oi the substances assayed before radiation: Rosmarinic acid
203

6. Posters
(RO)=Camosic acid (CA)=Oiosrrún (O}=Apigenin (AP)=O­
toeopherol (E)=Carnosol (Cl)=L_acid aseorbic (C)=Amifostine
(AMIF) (p<O.OOl»Green Tea Exh'act (GTA)-Rutín (R)=DimethyI
sulfoxide (DMSO) (p<O.OS»irradiated eontrols (Ci), with a
maximumradiation Protection Factorof 58%. When the substances
were administered irnmediately after radiation, the results were
different: CA=AP=E=Cl (p>O.OOl»O=C (p>o.05»Ci, with a
maximum protection factor oí 46%, while RO, At1IF, R and DMSO
had no protective capacity.
Cel1 survival obtained with 20 ~M and 40 ~M carnosic acid
administered before radiation with up to 10 Gy showed a
Protection Factor oí 100%, eliminatll1g 30.8% oí radioll1duced
cell death in both ceH Unes assayed. In the case oi normal cells
(PNT2), the protective capacity followed tlús order CA=mixture
(AP+CA)=mixture (AP+RO) (p<O.OOl»API=RO (p<O.05»Ci.
In the tumoral cell lineo a' ayed (B16FlO) on.ly CA acted a
radioprotector (p<O.OOl), while the rest oí the substances showed
nosignificanteffectin this respect (APIG, RO, DMSO and mixtures).
Carnosic acid shoved the greatest pre-exposure protective
effect, a good post-treahnent protection capacity and no toxicity
towards ceUs. For this reason, oUJ' results indicate that camosic
acid may be developed as a radioprotectant for humans against
the potentiaUy damaging effects of exposlIre to radiation.
Tbis report was supported by a grant from the National Spanish
R + O Programme CENIT oí the Spanish Ministry of Science and
Technology (acronym: SENIFOOD) and by a Fellowship oí the
International Atomic Energy Agency (GHAI0021).
TOPIes: 9. Trace Elements and Human Health
204

7. CARNOSIC ACID: A PROMISING MOLECULE
FOR RADIATION PROTECTION
(1)MiguelAlcaraz; (4)Julián Castillo; (4)Obdulio Benavente García; (2)David
Armero; (1)Miguel Alcaraz-Saura; (5)(*)Daniel Achel and (3)Vicente Vicente
Departments of (1)Radiology and Physical Medicine, (2)Nursing and (3)Pathology, University of Murcia. 30100-Campus de Espinardo. Murcia.
(4)Department I+D+ i Nutrafur S.A., Camino Viejo de Pliego s/n 30320-Alcantarilla, Murcia.
(5)Applied Radiation Biology Centre, Radiological and Medical Sciences Research Institute, Ghana Atomic Energy Commission, Legon-Accra, Ghana.
INTRODUCTION
There is a continued quest for the development of non-toxic compounds that can reduce the effect of exposure to
GENOTOXICITY
ionizing radiation. Such compounds could potentially protect humans against the genetic damage, mutation, alteration in the
human system and teratogenic effects of toxic agents (including radiation) which act through the generation of free radicals.
For many years, and despite their high toxicity, only sulfhydryl (SH) compounds were known to provide radioprotective effects
when administered biologically before exposure to ionizing radiation and only recently were other radioprotective substances
discovered whose common characteristic was their antioxidant capacity. Subsequently, other substances were found to have
the same effect when administered after exposure to ionizing radiation and, more recently, some substance have been
MN/500 CB
described as radioprotectors only when administered after exposure to radiation. Yet other substances present a
radioprotective capacity that differs according to whether they are administered pre or post radiation. Fig. 1: Different types of cells in the
In this work we assayed different antioxidant compounds that show protective capacity against the chromosome damage microscopic preparations: CB:cytokinesis-
induced by ionising radiation and related their effects with their chemical characteristics (Alcaraz et al., 2009) blocked cell; MNCB: cytokinesis-blocked cell
with micronúcleos; L: lymphocyte; E:
erythrocyte hemolized.
MATERIAL AND METHODS
Treatments
Chemicals: 82% Carnosic acid (oil soluble, os) (CA), 86% carnosol (os) (COL), green tea extract (90% catechins) (p<0,001) versus C
(water soluble, ws) (GTEA) and grape seed extract (95% procyanidins) (ws) (GSE), were supplied by Nutrafur S.A. CYTOTOXICITY (p<0,001) versus Ci
(Murcia, Spain). 95% rosmarinic acid (ws) (RO), 99% L-ascorbic acid (ws) (AA) and 99% δ-tocopherol (os) (E) were o (p<0,001) versus Ci
obtained from Sigma Co. (Madrid, Spain). Dimethylsulphoxyde (DMSO) was obtained from Merck (Darmstadt,
60
Germany). Dimethyl sulphoxide (DMSO) and Rutin (R) were obtained from Merck (Darmstadt, Germany); and 55
Magnitude of protection (%)
Amifostine (AMF) were obtained from Schering-Plough, SA (Ethyol® injectable, Madrid, Spain). CA was studied by 50
Magnitude of Protection (%)
means of two routine assays commonly employed in radiological protection: 45
Cellular survival (%)
40
Genoprotection assay: We used the cytokinesis-block micronucleus test (MN) with cytochalasin-B on irradiated 35
human lymphocytes to evaluate the genoprotective effects of these substances and CA following the technique 30
described by IAEA (2001). Samples of blood were cultivated at 37°C for 72 hours in 4.5 ml of HAM F-10 medium 25
20
(Sigma Co, Madrid) containing 15% fetal bovine serum, 16µg/ml phytohemaglutinin (Sigma Co, Madrid), 100 µg/ml 15
(penicilin/streptomycin) penicillin/streptomycin (Sigma Co, Madrid) and glutamine 1 µg/ml; cytochalasina B Apigenin-K 24h
10
(6µg/ml, Sigma Co.) was added 44 hours after the inception of the cultivation. At the end of 72 hours the culture Doses
5
0
was terminated and the cells harvested. At harvest the culture solution was centrifuged and a hypotonic solution RO CA E AP AA Amif Te R D DMS
KCl (75μM) was added to the collected cells. A fixative solution of methanol:acetic acid (3:1) was added and slides PF (%) 57.7
PF (%) 57,7 50
50 50
50 50
50 38.5
38,5 38.5
38,5 23.1
23,1 23.1
23,1 23.1
23,1 16.6
16,6
prepared. The preparations were stained with May-Grünwald and Giemsa stains. Scoring was done by counting the
Cellular survival (%)
Trratamientos
Treatments
Treatments
number of MN per 3000 binucleated cells (CB), using a Zeiss light microscope (Oberkochem, Germany) with a
magnification of 400X. The test substances were added to the samples of human blood cultures to obtain a final
concentration of 25 μM, before and after the exposure to X-rays.
Cytoprotection assay : We used the MTT cell survival assay to assess cytoprotective effect using two cell lines
PNT2 (normal epithelial cells) and B16F10 (melanoma) cell lines. The cells were cultivated normally in the presence
of CA and other test compounds (20μM and 40μM) for 24 and 48 hr after the exposure to X-rays. Briefly: the cells Doses
were incubated in 200 μl of medium supplemented with 50 μl of MMT (MTT) (8mg/ml) in 96 well microplates (4h,
37ºC, 5%CO2). After centrifugation (240g for 10 mn) to remove non metabolized MTT, 100 μl of DMSO was added to
Fig. 3: Control+Carnosic acid 24h and
each well to solubilized the MTT taken up by the live cells. Finally, MTT reduction data were expressed as
48h (PNT2 cell line)
absorbance at 570nm and 690nm with a Multiskan MCC/340P spectrophotometer. Fig. 2: Different types of cells in the microscopic
preparations: CB:cytokinesis-blocked cell; MNCB:
An X-ray equipment Andrex SMART 200E was used (YXLON International, Hamburg, Germany) operating at 120 kV, cytokinesis-blocked cell with micronúcleos; L:
4.5 mA, DFO 36 cm and ambient temperature to administer a dose of 2 Gy for the MN assay and 0 Gy, 4Gy 6Gy 8Gy lymphocyte.
and 10 Gy for the cell survival determinations. Statistical analysis consisted of pair wise contrast analysis of
variance to compare the percentages of cell survival in the cultures with different concentrations of the
compounds and contrasting their means with the method of minimum significant differences. The analysis was
Cellular survival (%)
Cellular survival (%)
carried out with a logarithmical transformation of the data to accommodate the adjustments made for the ANOVA.
Finally, the results were used to obtain the Magnitude of Protection: Magnitude of protection (%) = ((Fcontrol – Ftreated)
/ Fcontrol) x 100 (where Fcontrol = frequency of MN in irradiated blood lymphocytes and Ftreated = frequency of MN in
blood lymphocytes treated pre and post-γ-irradiation as described previously) and the Dose Reduction Factor
(DRF) determined.
RESULTS
Doses Doses
Fig. 4: Control+Carnosic acid 24h and
48h (B16F10 melanoma cells)
Genoprotection assay: The results point to a significant genoprotective capacity of the
substances assayed before radiation: Rosmarinic acid (RO)=Carnosic acid (CA)=Diosmin
(D)=Apigenin (AP)=δ-tocopherol (E)=Carnosol (Cl)=L_acid ascorbic (C)=Amifostine
Cellular survival (%)
Cellular survival (%)
(AMIF) (p<0.001)>Green Tea Extract (GTA)=Rutin (R)=Dimethyl sulfoxide (DMSO)
(p<0.05)>irradiated controls (Ci), with a maximum radiation Protection Factor of 58%.
When the substances were administered immediately after radiation, the results were
different: CA=AP=E=Cl (p>0.001)>D=C (p>0.05)>Ci, with a maximum protection factor of
46%, while RO, AMIF, R and DMSO had no protective capacity.
Cytoprotection assay : Cell survival obtained with 20 µM and 40 µM carnosic acid Doses Doses
administered before radiation with up to 10 Gy showed a Protection Factor of 100%,
eliminating 30.8% of radioinduced cell death in both cell lines assayed. In the case of
normal cells (PNT2), the protective capacity followed this order; CA=mixture
(AP+CA)=mixture (AP+RO) (p<0.001)>API=RO (p<0.05)>Ci. In the tumoral cell lines
Cellular survival (%)
Cellular survival (%)
assayed (B16F10) only CA acted as radioprotector (p<0.001), while the rest of the
substances showed no significant effect in this respect (APIG, RO, DMSO and mixtures).
CONCLUSION Doses Doses
Carnosic acid showed the greatest pre-exposure protective effect, a good post-treatment protection
capacity and no toxicity towards cells. For this reason, our results indicate that carnosic acid may be Figure 5: Cellular survival (%) with different mixtures of substances. (p<0.001 versus Ci).
developed as a radioprotectant for humans against the potentially damaging effects of exposure to
radiation.
This report was supported by a grant from the National Spanish R + D Programme CENIT of the Spanish Ministry of Science and
Technology (acronym: SENIFOOD) and by a Fellowship of the International Atomic Energy Agency (GHA10021).