Spontaneous sister chromatid exchanges in mitotic chromosomes of cattle (Bos taurus L)

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Spontaneous sister chromatid exchanges in mitotic

chromosomes of cattle (Bos taurus L)

D Di Berardino, Mb Lioi, Mr Scarfi, V Jovino, P Marigliano

To cite this version:

Original

article

Spontaneous

sister

chromatid

_exchanges

in mitotic

chromosomes

of

cattle

(Bos

taurus

_L)

D Di

Berardino

MB Lioi

MR

Scarfi

3

V Jovino

1

P

_Marigliano

1

University

of Naples

‘Federico

II’, Department of

Animal

_Science,

80055

Portici,

Naples;

2

University of Basilicata, Department of

Animal

_Science,

85100

_Potenza;

3

CNR-IRECE,

80124 Naples, Italy

(Received

9

_{September 1994; accepted}

12

_May

₁₉₉₅₎

Summary -

Peripheral

blood

lymphocytes

from 4 cattle donors were _grown for 2 cell

cycles

in the presence of 0.1, 0.25, 0.5, 1.0, 2.5 and 5.0

_wg/ml

of

_{5-bromodeoxyuridine}

in order to detect the _spontaneous level of sister chromatid

_exchanges

and the

_{dose-response}

relationships

at low

_{bromodeoxyuridine}

levels. Sister chromatid

_exchanges

were counted

on 50 s

_{cycle metaphase spreads,}

_{randomly scored,}

for each donor at each dose. The mean

rate of sister chromatid

_{exchanges/cell}

at 0.1

_!Lg/ml

of BUdR was 2.48 f

_1.75,

which can

be considered as _spontaneous. The

dose-response

curve increased more

_rapidly

from 0.1 to

0.5

_!g/ml

of

_{bromodeoxyuridine}

and less

_rapidly

at

_higher

concentrations. The

_frequency

distribution of sister chromatid

_{exchanges/cell}

and that of chromosomes

_showing

various numbers of

_exchanges

followed the Poisson

_probability

function

only

within the range of 0.1 to 1.0

_wg/ml

of

bromodeoxyuridine.

The usefulness of

determining

the spontaneous level of sister chromatid

_exchanges

in domestic animals is discussed in relation to its

_possible

application

for selection _programs.

spontaneous sister chromatid _exchange

_/

SCE

_/

_lymphocyte culture

_/

cattle

Résumé - Estimation des _{échanges interchromatidiques spontanés} dans les

chro-mosomes _mitotiques de bovin

(Bos

taurus

_L).

Des cultures de

_lymphocytes

obtenus à partir du sang

périphérique de

4 donneurs

bovins ont été réalisées

_pendant

₂

cy-cles cellulaires. Ces cultures sont

_effectuées

en

_présence

de

_{0,10, 0,25, 0,50, 1,0, 2,5}

et

5,0

J

.Lg/ml

de

_{bromodéoxyuridine}

_(BrdU).

Nous avons étudié les relations

_réponse

(échanges

chromatidiques)-

dose de BrdU

_afin,

notamment, de connaître le niveau

_spontané

des

échanges chromatidiques

chez cette

espèce.

Ces

_échanges

ont été

comptés

sur 50

plaques

en

_métaphase

II

_(par

donneur et par

_dose)

choisis par hasard. Le taux moyen

d’échanges

rapide augmentation

entre

0,10

et

_0,50pg/ml

de BrdU. Elle est

_{plus rapide}

_{que pour} des-concentrations en BrdU

plus

élevées. Les distributions de

fréquence

des SCE et des

chro-mosomes _portant un nombre variable

_{d’échanges}

suivent une loi de Poisson seulement pour des concentrations de Brd U de

0,10

à

_1,00J.

_L

_g/ml.

L’utilité de déterminer le niveau spon-tané des

_{échanges chromosomiques}

chez les animaux

_domestiques,

_pour une

_application

possible

aux _programmes de

_sélection,

est discutée.

échange interchromatidique spontané

/

culture de lymphocytes

/

bovin

INTRODUCTION

Spontaneous

sister chromatid

_exchanges

_(SCEs)

were first demonstrated

_by

Kato

(1974)

and Wolff and

_Perry

₍₁₉₇₄₎

in Chinese hamster cell lines.

_{Subsequently,}

other authors have extended these studies in in vitro

_(Tice

et

_al,

₁₉₇₆₎

and in vitro

systems

(Mazrimas

and

Stetka, 1978; Tsuji

and

_{Kato, 1981; Morgan}

et

_al,

_1986).

It is now well established that

_spontaneous

SCEs occur as an

_integral

_part

of

DNA

_replication,

even in the absence of

_agents

known to induce SCEs

_(Tucker

et

_al,

1986),

and data are available for several

_species,

including

man, and various cell lines.

In domestic

animals,

SCEs have been studied in several

species

of cattle

_(Di

Berardino and

_{Shoffner, 1979;}

McFee and

_{Sherrill, 1979;}

Di Berardino et

_{al, 1983;}

Iannuzzi et

_{al, 1991a, b;}

Catalan et

_al,

₁₉₉₄₎

_pigs

and

_sheep

_(McFee

and

_Sherrill,

1979),

goats

(Sanchez

and

_Burguete,

_1992;

Di Meo et

_al,

₁₉₉₃₎

and river buffalo

(Iannuzzi

et

_al,

_1988).

In the

majority

of these

_studies,

_{bromodeoxyuridine}

_(BUdR)

was utilized at

a final concentration of 10

!g/ml.

Since BUdR is itself an inducer of SCE

_(Latt,

1973;

Kligerman

et

_al,

_1982),

the mean rates of

_SCE/cell

detected so far reflect

mostly

induced

_exchanges.

At the

moment,

nothing

is known about the

_proportion

of

_spontaneous

versus induced SCEs.

For this _reason, we decided to re-examine the SCE data obtained in domestic animals

_by

_focusing

our attention on the

_spontaneous

yield

of SCEs which also

provides

an indirect estimation of the BUdR-induced SCEs.

The

_present

paper refers to the

spontaneous

rate of SCEs in cattle

_(Bos

taurus

_L).

MATERIALS AND METHODS

Peripheral

blood

_samples

were obtained from 4

(2

males and 2

females)

_clinically

healthy,

unrelated,

cattle of the Italian Friesian breed

_nearly

6 months of _age. From each

animal,

0.5 ml

_aliquots

of whole blood

₍₃

x

10

6

_lymphocytes)

were

added to each of 6 culture flasks

_containing

9.5 ml of RPMI 1640 medium

_(Gibco,

Dutch

_modification

New

_York,

_USA)

_together

with 1 ml of fetal calf serum

_(Gibco),

0.1 ml

of

_L

_-glutamine,

30

_!1

of

_{antibiotic/antimycotic}

mixture

_(Gibco)

and 0.1 ml

of Pokeweed

_mitogen

_(Gibco).

All cultures were _{grown at} 37.5 °C. After 36 h from

_initiation,

BUdR

_(Sigma,

Saint

_Louis,

MO,

USA)

was added to each culture

The cultures were

_protected

from

light

and allowed _{to grow} for an additional

36 h. Colcemid was added for the final 2 h. Harvested cells were treated with

hypotonic

solution

_(KCI,

0.075

_M)

for 20 min at 37.5 °C and fixed 3 times with

methanol/acetic

acid solution

_(3:1).

Air-dried slides were stained with a

0.2%

acridine orange solution in

_phosphate

buffer

_{(pH 7.0)}

for 10

_min,

washed

_thoroughly

in

_{tap water,}

mounted in

_phosphate

buffer and sealed with

_paraffin.

SCEs were

counted on 50 s

_{cycle metaphase spreads,}

_randomly

scored for each

_animal,

for each

BUdR level. To avoid

_possible

individual

_bias,

all

_scoring

was

_{performed by}

the same

person. In our

experimental

conditions it was not

_possible

to utilize BUdR doses lower than 0.1

_wg/ml

because of the

_poorly

defined sister chromatid differential.

RESULTS

Table I

_reports

mean number and standard deviation of the SCEs _per cell scored at each BUdR level in the 4 animals tested. At the lowest dose of 0.1

_!g/ml

of BUdR the individual

_SCE/cell

rates varied from 1.64 ! 1.16 to

3.62 t 2.1,

with an _average

of 2.48 f 1.75. At the dose of 5.0

_!g/ml

of

_BUdR,

the individual

_SCE/cell

rates varied from 3.64 f 1.9 to 6.58 !

_3.05,

with an _average of 5.16 ! 2.75. The

_analysis

of variance

_performed

on these data indicated

_significant

differences at each BUdR

level

_(P

<

_0.001)

among the 4 animals

investigated.

Figure

1 shows the mean rate of

_SCE/cell

in the 4 donors tested. The overall mean rate of

_SCE/cell

increased more

rapidly

between 0.1 and 0.5

!g/ml

of BUdR

and less

_rapidly

at further

_{concentrations,}

thus

_indicating

a saturation level. This

trend was found to be

_logistic

_(y

=

a/I

₊

e

bx

,

where a = 5.05 and

b = -1.31).

When the data were examined

_by

_using

_quartile

statistics

_(fig

_2),

50%

of the

SCE/cell

values observed between 0.1

_(D1)

and 0.25

_{(D2) !g/ml}

of BUdR remained

fairly

stable, starting

to rise at

_higher

concentrations. This would

suggest

that

(in

our

_laboratory

conditions)

_{up to} 0.25

!g/ml

of BUdR the

_exchanges

could be

considered as

_spontaneous,

_being

little affected

_by

the

_analogue.

In order to characterize the distribution of the

_exchanges

in the cell

_population,

table II was

_prepared,

in which the

SCE/cell

values of the 4 animals observed

distribution. The

_chi-square

_analysis

revealed

_that,

at the

5%

_probability

level,

the

_{expected frequencies}

were close to the observed

_only

within the _range of

0.1 to

_{1.0 !g/ml}

of

BUdR,

whereas at

_higher

BUdR concentrations the observed distribution deviated

_{significantly}

from the theoretical model. Such a deviation

!g/ml

of BUdR and the Poisson

_expected

_frequencies.

It is

_quite

evident that in D6 the Poisson

_expectations

do not fit the observed

_frequencies

₂

_X

₍

= 30.2

>

X5

.

05

)

’

However,

when 2

_{subpopulations}

of

_lymphocytes

are considered instead of 1

_(Exp.

D6a

_line),

the Poisson distribution fits well the observed

_frequencies,

with a

chi-square of 13.33

(x)

05

=

18.3;

df

=

10).

Table III

reports

the overall number of chromosomes with

0,

1 and 2 or more

exchanges

at each BUdR level and the number

_expected

on the basis of the Poisson

distribution. The

_{chi-square analysis}

revealed that from 0.1 to 1.0

_wg/ml

of BUdR the

_{expected frequencies}

were close

_(P

<

_0.05)

to the

_observed,

whereas at

_higher

concentrations

_they

were

not,

thus

_confirming

to a

large

extent that low BUdR levels

_provide

a better fit to the Poisson

expectations,

as

_{previously suggested}

_by

Tucker et

_{al (1986).}

DISCUSSION

Spontaneous

SCEs

_provide

an indication of the extent of somatic recombination

has never been

_attempted.

The

_only

_report

available is that of McFee and Sherrill

(1979)

who detected a mean value of 5.95

SCE/cell

in cattle

_{lymphocytes exposed}

to 0.5

_!g/ml

of BUdR. The

_present

paper

reports

a mean value of 2.48

SCEs/cell

at a

was not

_satisfactory

for SCE detection. Even

_though

BUdR concentrations less than 0.1

_!g/ml

could be used in other

laboratory

conditions

_(Tucker

et

_al,

_1986),

the mean value of 2.48

SCEs/cell

can be considered _very close to the

spontaneous

yield

of SCEs in cattle. Since the

_exchange

_frequency

observed is the sum of

exchanges

formed

_during

2

_subsequent

cell

_cycles,

the _average

_frequency

of SCEs per cell

generation

is 1.24.

_By

_considering

that cattle somatic cells have a

_diploid

number of 60 and a

_diploid

DNA content of 6.4 pg

(Green

and

Bahr,

1975),

the

corresponding

values are 0.02 SCEs per chromosome per cell

generation

and 0.19 SCEs _per

_picogram

of DNA.

The

_spontaneous

_yield

of 2.48

_SCEs/cell

in cattle chromosomes is _very close to that

reported

by

Kato

₍₁₉₇₄₎

in a

_{pseudodiploid}

Chinese hamster cell line

exposed

to the same BUdR concentration

(2.3 SCEs/cell)

but

considerably

lower than that

reported by

Tucker et al

₍₁₉₈₆₎

in human and mouse blood

_lymphocytes

_exposed

to 30 nM of BUdR

_(7.2

and 4.9

_{SCEs/cell, respectively).}

McFee and Sherrill

₍₁₉₇₉₎

also

reported higher

values of

_SCEs/cell

in

_{cattle, pig,}

_sheep

and human

_lymphocytes

but

_they

used 0.5

_wg/ml

of BUdR. The

_{dose-response}

curve of cattle chromosomes

exposed

to

_increasing

doses of BUdR was found to be

_logistic.

The

_yield

of SCEs

rises

_quite

_steeply

between 0.1 and 1.0

_wg/ml

of BUdR. Above this concentration the curve rises

_{slowly, indicating}

a saturation level. This

pattern

is very similar to that found

_by

Wolff and

_Perry

₍₁₉₇₄₎

in Chinese hamster ovary cells grown under BUdR concentrations

_varying

from 0.25 to 20

_!g/ml,

and also to that

_{reported by}

McFee and Sherrill

₍₁₉₇₉₎

on cattle

lymphocytes

_grown at concentrations

_varying

from 0.5 to 20

_!g/ml

of BUdR.

However,

a different

pattern

of the

_{dose-response}

curve has been

reported by

other authors who found a

_plateau

of the SCE

_frequencies

at low BUdR doses in

a Chinese hamster

pseudodiploid

cell line

(Kato, 1974),

in an in vivo rat

_system

(Tice

et

_al,

_1976),

in HeLa cells

_(Tsuji

and

_Kato,

_1981),

and in human and mouse

lymphocytes

(Tucker

et

_al,

_1986).

These

_{contradictory}

results can be accounted for

_by

a

_variety

of factors such as

_species,

individuals,

cell source and

laboratory

conditions

_(Das

and

_Sharma,

_1983).

_However,

when

_quartile

statistics were

_applied

to our

_data,

a

_{fairly steady}

line was found between 0.1 and 0.25

_!g/ml

of

BUdR,

thus

_indicating

that

50%

of the cells scored were little affected in their SCE _response

by

the increase of BUdR.

_Furthermore,

when the data

_reported

in table I are

examined within the range from 0.1 to 1.0

_wg/ml

of

_BUdR,

donors n3 and n4 show a

plateau

of SCE

_values,

while donors nl and n2

_display

a

_rapid

increase of

_SCE/cell

frequencies.

This

_finding

indicates that individual variations

_play

an

_important

role in

_determining

the overall

_{dose-response}

_pattern.

Our data also indicate that when Pokeweed is used as

_mitogen

for SCE

_studies,

BUdR concentrations

_higher

than

1.0

_!g/ml

_may

_provide

SCE responses which do not follow Poisson

_{expectations,}

due to the _presence of 2

_{subpopulations}

of B and T

_lymphocytes

which are known

to differ in their SCE _response,

_{proliferation}

rate and

_sensitivity

to

_chemically

induced

_damage

_(Lindblad

and

_{Lambert, 1981;}

Erexson et

_{al, 1983;}

Bloom et

_al,

1993).

Recently,

Catalan et al

₍₁₉₉₄₎

studied a _group of 24 cattle of different

breeds,

ages and

farms,

and

_reported

a

_spontaneous

incidence of 5.77 ! 0.082

(se)

SCEs

_by

using

5

_R

_g/ml

of BUdR. While this value is in close

_agreement

with ours

(5.16!2.75

(sd) SCEs/cell),

we can

_hardly

believe that SCEs obtained at 5

_!g/ml

of BUdR can

The results of the

present

investigation

demonstrated that in cattle the mean

rates of

_SCE/cell

_reported

_by

various authors so far include a

_spontaneous

level of

2.5

_SCEs/cell,

the

_remaining

SCEs

_being

induced

_by

BUdR.

The SCE test is

_commonly

used to assess chromosome

_instability

_(Chaganti

et

al,

1974)

and

_genetic

_damage

under

_mutagenic

exposure

(Carrano

et

al,

1978).

Spontaneous

SCEs have been related to the ’unscheduled’ DNA

_synthesis

_occurring

in mammalian cells to

_{repair apurinic}

DNA sites

_(Verly

et

_al,

1973; Kato,

1974).

Even

_though

the molecular mechanism of SCE formation still remains to be

elucidated,

both

_types

of SCEs seem to be related to the DNA

_{repair activity}

of

the cells. It seems,

therefore, important

to ascertain the

_proportion

of

_spontaneous

versus induced

_SCEs,

rather than the overall SCE _response alone. Variations

among

species, breeds,

and individuals in the

_proportion

of

_{spontaneous/induced}

SCEs would reflect different DNA

_repairing

efficiencies which should be taken into consideration when

breeding animals,

especially

those destined for artificial

insemination,

are evaluated and selected for animal

_{production improvement.}

This

aspect,

however,

is

_worthy

of further

_{investigation.}

ACKNOWLEDGMENTS

Special

thanks are

_given

to C Vitale of the

_Department

of

_{Statistics, University}

of

Salerno,

for his valuable collaboration in the statistical

_analysis.

This research was

supported by

the National Research Council of

_Italy,

_Special

_{Project Raisa, sub-project}

No 3

Paper

No 2234.

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