Mutagenesis vol. 22 no. 5 pp. 317–320, 2007
Advance Access Publication 18 June 2007
doi:10.1093/mutage/gem019
Chromosome fragility in cattle with chronic enzootic haematuria
V. Peretti*, F. Ciotola1, S. Albarella, V. Russo2, G. P. Di
Meo3, L. Iannuzzi3, F. Roperto2 and V. Barbieri
Department of Animal Science and Food Inspection, Faculty of Veterinary
Medicine, University Federico II, Naples, Italy, 1Department of Experimental
and Clinical Medicine, University of Catanzaro ‘‘Magna Graecia’’, Catanzaro,
Italy, 2Department of Pathology and Animal Health, University Federico II,
Naples, Italy and 3CNR-ISPAAM, Laboratory of Animal Cytogenetics and
Gene Mapping, Naples, Italy
Chronic enzootic haematuria (CEH) is a severe syndrome
due to prolonged ingestion of toxic principles of bracken
fern, such as quercetin and ptaquiloside. Little information
is available on chromosomal instability of cattle with access
to bracken fern and suffering from CEH. In the present
study, 45 cattle, aged from 7 to 12 years and pastured in
the south of Italy, were cytogenetically investigated for the
first time in search of both chromosomal aberrations
(aneuploidy, gaps, chromatid breaks, chromosome breaks
and fragments) and sister chromatid exchanges (SCEs). Of
these animals, 30 (group 1) had access to bracken fern and
showed signs of CEH, and 15 (group 2; control) did not.
Percentage of abnormal cells (aneuploidy, chromatid
breaks, chromosome breaks and fragments) was higher
in animals affected by CEH (34.7%, group 1) than that
(24.3%) reached in the control (group 2). The same results
were achieved when including gaps. Indeed, the mean
number of cells with structural aberrations excluding gaps
(chromatid breaks, chromosome breaks and fragments)
per cell was higher (P < 0.001) in animals affected by CEH
(0.16 0.36) than that (0.09 0.29) found in the control.
Chromosome fragility in cells of animals affected by CEH
was also confirmed when applying the SCE test: statistically higher levels (P < 0.001) of SCEs were observed in
animals with CEH (7.35 3.59 SCE/cell, group 1) than
those in the control (5.40 2.68 SCE/cell).
Introduction
Chronic enzootic haematuria (CEH) is a severe syndrome due
to prolonged ingestion of bracken fern (Pteridium aquilinum),
the only plant proven to cause cancer in animals (1). This
syndrome occurs in several areas worldwide and is very
common in southern Italy where bracken is widespread (2).
Pinto et al. (3) in an epidemiological study demonstrated an
association between CEH and the level of pasture infestation
with bracken fern. It is well known that this plant contains toxic
compounds like ptaquiloside (PT) and quercetin, which have
mutagenic and carcinogenic effects, mainly in the bladder (2).
PT is a sesquiterpenoid glycoside capable of inducing clastogenesis in cell cultures and also with mutagenic and
carcinogenic activity (1,4). Shahin et al. (5) proposed a PT
cancer model to explain bracken-induced carcinogenesis and
supposed that it occurs at cellular and molecular levels. At the
molecular level, PT is converted into a conjugated dienone
(APT) in alkaline conditions, which then alkylates DNA at N3
of adenine within 24 h of exposure (6). At the cellular level,
affected cells have the capacity to repair such lesions in a short
period of time, but a few of these lesions lead to erroneous
repair which culminates in mutations in key genes, initiating
cancer. Before any histopathological signs of neoplasia could
be observed, mutations are detected in H-ras, an oncogene
associated with several types of cancer including those
occurring in colon, urinary bladder and mammary glands.
Sardon et al. (7) confirmed the presence of DNA changes in
bladder lesions of cattle exposed to bracken fern, H-ras
mutations being significantly increased in chronic cystitis and
tumours. The role of quercetin remains ill defined (5).
A strong relationship between bovine papillomavirus (BPV)2 and bracken fern in both experimental and naturally
occurring bovine bladder cancer has been established (8–10).
Although the synergism between the virus and bracken is still
poorly understood, it is likely that BPV-2 infects the bladder
mucosa, producing an abortive latent infection, and the latent
BPV is activated by bracken-induced immunosuppression, thus
initiating progression to malignancy (10).
Chromosome aberrations, especially chromatid breaks, are
also known to occur in humans suffering from cancer
(reviewed in ref. 11). There is little information on chromosomal instability in cattle with access to bracken fern and
suffering from CEH. More recently, Lioi et al. (12) observed
an increase in the number of structural chromosomal aberrations in 56 cattle with CEH raised on pastures giving access to
bracken fern with the highest clastogenic effect observed in
cattle with urinary bladder cancer (27 animals). However, these
authors did not investigate for the presence of aneuploid cells
and did not use the sister chromatid exchange (SCE) test.
The SCE test has been used to detect genome stability in
humans (13) and in the main livestock species such as cattle
(14–16), river buffalo (17), goat (18), sheep (19) and pig
(20,21), as well as to discover DNA damage caused by
a variety of natural and artificial chemical compounds (22–24).
In the present study, we report the results of our investigation
on chromosome stability of a representative sample of cattle
with CEH using for the first time cytogenetic tests that detect
both chromosomal aberrations (aneuploidy, gaps, chromatid
breaks, chromosome breaks and fragments) and SCEs.
Materials and methods
Animals
The study was performed on 45 cattle, aged from 7 to 12 years and naturally
pastured in southern Italy. Of these, 30 (group 1) had access to bracken fern and
15 (group 2; control) did not. Animals from group 1 showing signs of CEH
*To whom correspondence should be addressed. Tel: þ39 081 2536433; Fax: þ39 081 292981; Email: [email protected]
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V. Peretti et al.
were monitored during life and studied after slaughter at an abattoir. All
animals from group 2 were healthy and did not show signs of CEH.
The control group animals were selected from those with the same age as
animals affected by CEH (from 7 to 12 years old) because cytogenetic studies
performed in humans (25) and animals (20) report a relationship between the
increase in SCEs and age.
Clinical and histological evaluation
CEH (group 1) was diagnosed on the basis of clinical history or clinical
examination. The neoplastic lesions at the urinary bladder were detected by
gross examination (Figure 1). Tumour samples were fixed in 10% formalin,
embedded in paraffin wax and stained with haematoxylin and eosin for routine
histological examination. The following microscopic patterns were observed in
the animals: inflammation (2 animals), dysplasia (1 animal), vascular cancer (6
animals), carcinoma in situ (7 animals), and carcinoma (14 animals).
Cytogenetic analysis
Peripheral blood (1 ml) was cultured in RPMI medium, enriched with foetal
calf serum (10%), l-glutamine (1%) and Concanavalin A (1.5%) (as mitogen)
for 72 h at 37.8°C. Two types of cell cultures, without (normal cultures) and
with addition of 5-bromodeoxyuridine (BrdU) (10 lg/ml) were performed. In
the latter, BrdU was added 30 h before harvesting to allow its incorporation into
DNA during the last two cell cycles (SCE test). Cells from both types of cell
cultures were harvested after Colcemid (0.3 lg/ml) treatment for 1 h and given
hypotonic treatment (KCl 0.5%) and three fixations in methanol–acetic acid
(3:1), the third overnight. Three drops of cell suspension were air-dried on
cleaned and wet slides which were stained a day later with acridine orange
(0.01% in a phosphate buffer, pH 7.0) for 10 min, washed in tap and distilled
water and mounted in the same phosphate buffer. Slides were observed 24 h
after staining or later (1 week).
At least 50 cells per animal were examined from slides of normal cultures to
detect aneuploidy and structural aberrations, i.e. gaps, chromatid breaks,
chromosome breaks and fragments (Figure 2) that were classified according to
the criteria suggested by Savage (26) and Carrano and Natarajan (27). For the
SCE test, 35 cells at the second cycle of replication (Figure 3) in the presence of
BrdU and with a complete chromosome set (2n 5 60) were considered. All
metaphases plates were observed under a fluorescence Nikon microscope,
captured with a digital camera Nikon DS U1, transferred onto a PC and later
processed by image analysis software of two cytogeneticists.
Fig. 2. Chromosome aberrations in cattle metaphases: a 5 gaps; b 5
chromatid breaks; c 5 chromosome breaks and d 5 fragments.
Statistical analysis
An independent two-sample Student’s t-test was performed and a confidence
interval generated to compare the two groups of animals (with and without
CEH), determining whether or not there is any evidence that the differences
between the three cytogenetic analyses we applied: aneuploidy, chromosome
abnormalities (gaps, chromatid breaks, chromosome breaks and fragments) and
SCE were other than 0.
Results
The percentage of abnormal cells (chromatid breaks, chromosome breaks and fragments) was higher in the animals with
CEH (16.1%, group 1) than that (9.7%) in the control. When
including aneuploidy and gaps, percentages of abnormal cells
were very high in both animals affected by CEH (92.6%, group
1) and the control (74.1%) (Table I), although percentages of
Fig. 3. Cattle female metaphase plate (2n 5 60, XX) treated for the SCE test
and stained with acridine orange. SCEs are indicated (arrows).
Table I. Percentages of abnormal (chromatid breaks, chromosome breaks and
fragments) cells in animals with CEH (group 1) and control (group 2) with
and without gaps and aneuploidy
Groups
Fig. 1. (A) Normal cattle urinary bladder. (B) Details of the cattle urinary
bladder showing various lesions due to CEH.
318
1 (30)
2 (15)
Cells
examined (n)
1500
750
Abnormal cells with
gaps and aneuploidy
Abnormal cells without
gaps and aneuploidy
n
%
n
%
1389
556
92.6
74.1
241
73
16.1
9.7
Chromosome fragility in cattle
Table II. Mean and SD of aneuploid cells, cells with structural aberrations excluding gaps, chromatid breaks, chromosome breaks, fragments and gaps in cells of
animals with CEH (group 1) and control (group 2)
Groups
Cells
examined (n)
Aneuploid cells
(mean/cell SD)
Cells with structural
aberrations excluding
gaps (mean/cell SD)
Chromatid breaks
(mean/cell SD)
Chromosome breaks
(mean/cell SD)
Fragments
(Mean/cell SD)
Gaps
(Mean/cell SD)
1 (30)
2 (15)
1500
750
0.21 0.41*
0.15 0.36
0.16 0.36**
0.09 0.29
0.12 0.36*
0.08 0.29
0.05 0.27***
0.02 0.16
0.02 0.16***
0.005 0.09
2.83 1.90**
1.71 1.49
*P , 0.01; **P , 0.001; ***P , 0.05.
Table III. Animals, cells studied and mean SCE values in cells of animals
with CEH (group 1) and control (group 2)
Groups
1
2
Number of animals
30
15
Cells examined (n)
1050
525
SCE/cell
n
Mean SD
7717
2825
7.35 3.59**
5.40 2.68
**P , 0.001.
abnormal cells still remain higher in animals affected by CEH
than those reached in the control.
Mean values of aneuploid cells (79% 58 , 2n , 60; 21% 50 ,
2n , 58) were, respectively, 0.21 0.41 in the animals with
CEH and 0.15 0.36 in the control group. The mean number
of abnormal cells without gaps was higher in animals affected
by CEH (0.16 0.36) than that (0.09 0.29) found in the
control (P , 0.001).
Considering all structural aberrations, mean values of gaps
are higher in cells of animals affected by CEH (2.83 1.90)
than those found in the control (1.71 1.49), and similar
results were obtained in chromatid breaks (0.12 0.36, 0.08 0.29, respectively), chromosome breaks (0.05 0.27, 0.02 0.16, respectively) and fragments (0.02 0.16, 0.005 0.09,
respectively) (Table II).
Significant increases (P , 0.001) in the mean number of
SCE/cell were observed in the group of animals with CEH
(7.35 3.59 SCE/cell), compared with that in the control (5.40 2.68 SCE/cell) (Table III).
values of aneuploid cells were high not only in the animals
affected by CEH (0.21 0.41) but also in the control (0.15 0.36) (Table II), as previously found in cells of sheep exposed
and unexposed (control) to dioxins (23,24). It is difficult to
establish whether this phenomenon is due to the presence of
high percentages of chromosomal abnormality which can give
rise to chromosome loss in subsequent cell division, or it is
simply due to technical problems occurring during chromosome treatments. Indeed, all aneuploid cells were hypoploid
(2n , 60). However, only now (use of image processing and
analysis) can we easily count the number of chromosomes for
each metaphase, compared to previous studies where aneuploid
cells were difficult to detect by using only microscope
observation, especially in cattle.
The SCE test (Table III) confirmed chromosome fragility in
cells of animals affected by CEH when compared with the
control. Indeed, a significantly higher mean number of SCE/
cell was found in animals affected by CEH (7.35 3.59 SCE/
cell), compared with that of the control (5.40 2.68 SCE/cell).
Our study also showed that animals, especially grazing cattle,
may be used as environmental biological indicators of natural
toxic compounds by using cytogenetic tests, such as those
applied in the present study or, more generally, using biomarkers
of DNA damage. Such tests can measure the biological effects of
natural toxic compounds before overt disease develops.
Acknowledgements
This work was supported by grants from PRIN 2005.
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Prolonged ingestion of toxic principles in bracken fern, such as
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Received on December 21, 2006; revised on February 23, 2007;
accepted on April 5, 2007
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