Transcript Preliminary results on the genome instability evaluated by

M19.24
[email protected]
Preliminary results on the genome instability evaluated by Micronucleus Test in four Italian pigs Ancient
Autochthonous Genetic Types (AAGT)
Matassino, D.1,2, Falasca, D.1, Gigante, G.1, Varricchio, G.1, Fornataro, D.1
1ConSDABI.
NFP.I.-FAO- Centro di Scienza Omica per la Qualità e per l'Eccellenza Nutrizionali, Località Piano Cappelle-82100 Benevento, Italy,
2Dipartimento di Scienze biologiche e ambientali - Università degli Studi del Sannio, Via Port'Arsa,11-82100 Benevento, Italy
1.INTRODUCTION
The ‘genome instability’, both natural (aging) and induced by physical and/or chemical and/or environmental agents, may determine the loss of some portions of genome, probably due to
alterations of repair mechanisms. Therefore, the ‘genome instability’ could promote mutations, rearrangements, deletions and subsequent gene activations or inactivations of ‘polypeptide coding
DNA segments’ (‘genes’) and hence changes in the gene expression regulation mechanisms (Franceschi, 1994). Part of aberrations (generally ‘symmetrical events’ or ‘unstable aberrations’)
originates chromosome fragments without organelles for mitotic fuse anchoring (kinetochore and centromere), called ‘acentric fragments’ (AF). During cell division, some of these fragments
are excluded from major nuclei of daughter cells and produce small external nuclei (‘micronuclei’, MN), within the cytoplasm, singularly or together other fragments. Some MN may appear in
the cytoplasm of one or both daughter cells. According to the origin of the ‘excluded’ fragments, both or only one of the daughter cells undergo/oes a genetic loss: as mechanical separation
(‘bridges’) or fragment loss at anaphase.
These events will cause ‘cell death’, although‘cell death’(intended as end of division) could not arrive before 2÷4 divisions (Yamamoto e Kikuchi,
1980; Högsted e Karlsson, 1985; Preston et al., 1987; Degrassi e Tanzanella, 1988; Majone et al., 1990; Vanparys et al., 1990; Antoccia et al., 1990;
Kallio et al., 1993; Chen et al., 1994; Hayashi et al., 1994; Modesti et al., 1995; Nath et al., 1995). Furthermore, if an acute dose of radiations or a
short treatment with clastogenic agents are administered, the micronuclei production ends and in the new cells the frequency comes back at control
levels (not treated cells). Today one type of MN is known, but several types of aberrations may contribute to form them. Fig. 1 synthetizes the major
types of aberrations producing AF, including aberrations which give compound fragments, for example formed by segments deriving from more
chromosomes/chromatids or inducing mechanical separation problems (‘bridges’) at anaphase. In particular, it is possible to evidence in a MN the
presence of whole chromosome ( 1st type) or chromosomal fragments (2nd type); aneugenic agents (colchicin, vinblastine, sulfunate, vincristine,
ecc.), interfering with microtubule organization, may lead to formation of type 1 MN, while, the use of clastogenic agents (cyclophosphamide,
mitomicine C, ecc.), inducing chromatide breakage, may induce type 2 MN.
FIG. I. Main types of aberrations producing AF.
2. AIM
To estimate ‘genome instability’ of autochthonous genetic types (AGTs), especially ancient (AAGTs), in order to evaluate the possible damage of factors, with particular reference to the
environmental ones.
3. MATERIAL AND METHODS
Twelve pigs, three for each of the following AAGT: Calabrese (CL), Casertana (CT), Cinta Senese (CS) and Nero Siciliano (SC) were analysed using the technique reported by Matassino et al.
(1994) appropriately modified for pig. Peripheral blood was collected from femoral vein using heparinized vacutainers. Cell cultures were prepared with 8 ml of RPMI (Gibco), 1ml blood, 15 %
inactivated Foetal Calf Serum (FCS), 10 ml/ml L-glutammine and 10 ml/ml Pokeweed. After 44 hours of incubation, cytochalisin B was added to give final concentration of 6 mg/ml (Scarfì et al.,
1993). After 72 h of growth at 37 °C, cell suspension was treated with eritrocyte lysis buffer. After washing in RPMI 1640 (Gibco) supplemented with 2% FCS, cell suspension has undergone to
hypotonic solution for 15’. Slides were examined using a Leitz Diaplan microscope at 200 X magnification.
The statistical analysis was performed by chi square test.
4. RESULTS AND DISCUSSION
Table I shows the number of binucleated cells examined and in table II, number, percentage, mean, standard deviation and variation coefficient of binucleated cells with MN, independently from age
are reported.
As showed in graph I, in the limits of observation field, CL has a lower mean frequency of binucleated cells with MN in comparison with SC (P < 0.01; 2.06 vs 3.26) and CT (P < 0.005; 2.06 vs
3.43).
Finally, from table III, grouping the ages into three classes: 1. (< 30 months), 2. (30÷40 months); 3. (>40 months), it emerges that there is a trend to the increase of binucleated cells with micronuclei
from the first to the second class, while no appreciable change was observed passing from the second to the third age class.
The low number of subjects examined in the present paper doesn’t allow to make definitive conclusions; therefore, it is necessary to deepen knowledge of this phenomenon since that differences
among genetic types, especially autochthonous, within and inter species could be correlated to a different ‘constructivism capacity’ rather than adaptation to the different ‘microagroecosystems’ or
‘bioterritory’ ( Matassino, 1995).
TABLE I. Number (N) of examined binucleated cells and number and percentage of binucleated cells with
micronuclei (MN) within ancient autochthonous genetic type (AAGT).
SUBJECT
ANCIENT
AUTOCHTHONOUS
GENETIC TYPE
CL
CS
SC
CT
N
AGE (MM)
1
2
3
TOTAL
1
2
3
TOTAL
1
2
3
TOTAL
1
2
3
TOTAL
48
37
27
EXAMINED BINUCLEATED CELLS
WITH MN
N
N
c.v, %
533
17
3.18
833
15
1.80
1,157
20
1.70
2,523
52
2.24
667
21
3.14
815
18
2.20
1,034
34
3.28
2,516
73
2.88
695
20
2.87
849
28
3.29
969
34
3.50
2,513
82
3.23
937
45
4.80
996
33
3.31
1,035
24
2.31
2,968
102
3.49
72
30
20
62
32
32
50
36
36
GRAPH I. Percentage of binucleated cells with MN within AAGT, independently on age
(** = P< 0.01; ***= P < 0.005).
TABLE II. Number (N), mean ( x ), standard deviation (  )and percentage variation coefficient (c. v., %) of examined binucleated cells
with micronuclei (MN), within the ancient autochthonous genetic type.
SUBJECTS
EXAMINED BINUCLEATED CELLS
ANCIENT
AUTOCHTHONOUS
AGE (MONTHS)
WITH MN
N
N
GENETIC TYPE
xσ
x 
c. v., %
N
c. v., %
CL
3
37.3 ± 14.8
40
2,523
52
17.3 ± 2.5
15
CS
3
40.7 ± 36.8
90
2,516
73
24.3 ± 8.5
35
SC
3
42.0 ± 21.2
51
2,513
82
27.3 ± 7.0
26
CT
3
40.7 ± 9.9
24
2,968
102
34.0 ± 10.5
31
TABLE III. Number (N), mean ( x ), standard deviation ( σ )and variation coefficient (c.v., %) of examined binucleated cells
with micronuclei (MN) within age class, independently on AAGT.
AGE CLASS
(MONTHS)
SUBJECTS
N
N
EXAMINED BINUCLEATED CELLS
WITH MN
xσ
N
c. v., %
1.
(<30)
3
3,006
72
24±8.7
36
2.
(30÷40)
5
4,408
120
24.0±70
29
3.
(>40)
4
3,106
117
29.3±12.8
44
PERCENTAGE
(%)
4
3
2
1
0
CL
CS
SC
CE
AAGT
5. CONCLUSIONS
Genetic variability is the ‘conditio sine qua non’ to implement any genetic improvement programme. The extinction of the ‘autochthonous’ populations, especially ancient, leads to a loss of genetic
variability (up to today little known yet). In the future, this loss could negatively influence the possibility to have genetic information at disposal to improve the qualitative levels of ‘nutritional’ and
‘extranutritional’ biomolecules able to satisfy variations or innovations of human physiological needs due to the continuous changes in the microenvironment in which he is inserted. Variability of
these biomolecules from both qualitative and quantitative point of view is in relation to a higher or lower genetic variability.
Annual Meeting of the European Association for Animal Production (EAAP), Antalya (Turkey), September 17÷20, 2006