Differential sister chromatid exchange response in phytohemagglutinin and pokeweed stimulated lymphocytes of goat (Capra hircus L)

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Differential sister chromatid exchange response in

phytohemagglutinin and pokeweed stimulated

lymphocytes of goat (Capra hircus L)

D Di Berardino, V Jovino, A Crasto, Mb Lioi, Mr Scarfi, I Burguete

To cite this version:

Original

article

Differential sister chromatid

_exchange

response

in

phytohemagglutinin

and

pokeweed

stimulated

_lymphocytes

of

_goat

(Capra

hircus

_L)

D Di

Berardino

V Jovino

A Crasto

MB

Lioi

MR

Scarfi

I

_Burguete

!

Department of

Animal

_{Science, University of Naples}

’Federico

_II’,

80055

Portici,

Naples;

2

Department of

Animal

_{Production, University of Basilicata,}

Via N Sauro

_85,

85100

_Potenza;

3

CNR-IRECE, 80124 Naples, Italy

4

Department

of

Animal

_{Production, University of Murcia, Espinardo,}

30071 Murcia,

Spain

(Received

3 June

_{1996; accepted}

21

_January

₁₉₉₇₎

Summary - A differential sister chromatid

_{exchange (SCE)/cell}

response was observed

between

_{phytohemagglutinin}

_(PHA)

and

pokeweed

(PKW)

stimulated blood

lymphocytes

of goat

(Capra

hircus

_L)

exposed

to final concentrations of 0.1,

0.25,

0.5, 1, 2.5 and 5

_pg/mL

of BUdR. At 0.1

_!g/mL

of

BUdR,

the two

_mitogens

_{gave very} similar

_SCE/cell

responses: the SCE mean values were 3.17 ! 1.93 for PHA and 3.28 ! 1.76 for

PKW,

and the

_frequency

distributions fit very well the Poisson

probability

function with both

mitogens.

For 0.25

_v

_g/mL

and

increasing

BUdR

concentrations,

SCE/cell

rates for

_{pokeweed mitogen}

were

_{significantly higher}

than those of PHA. At 5

₄

_g/mL

of

BUdR,

the

_SCE/cell

response was 8.68 ! 3.24 for PKW and 6.96 ! 3.45 for

PHA,

and

the difference was

_{statistically significant}

_(P

=

_0.0001);

for both

_mitogens

the

SCE/cell

frequency

distributions fit the Poisson

_probability

function

_{only by adopting}

a Poisson ’mixture’

model,

which takes into account the presence of two different

subpopulations

of

cells.

sister chromatid

_exchange

_/

phytohemagglutinin

/

pokeweed

/

Poisson distribution

_/

goat

Résumé - Effets différentiels de la phytohémagglutinine et du phytolaque sur les échanges entre chromatides soeurs chez les _lymphocytes de la chèvre. Une

réponse

différentielle

pour le nombre

d’échanges

entre chromatides soeurs a été observée entre les

lymphocytes

de chèvre

_(Capra

Hircus

_L)

stimulés _par la

_{phytohémagglutinine (PHA)}

ou le

phytolaque (PKW),

après exposition

à des concentrations

_finales

de

0,1, 0,25, 0,5, 1, 2,

5

SCE/

cellules très similaire : les valeurs moyennes ont été de

3,17 ::!: 1, 93

pour PHA et

de 3, 28 !

1, 76

pour

PKW,

et les distributions de

_fréquences

se sont très bien

_ajustées

à une loi de Poisson _pour les deux

mitogënes.

À

partir de

_{0,25 J1g/mL,}

les

réponses

à PKW ont

_toujours

été

supérieures

à celles

correspondant

à PHA.

À

partir de 5

_J1

_g/mL

de

_BUdR,

la

_réponse

_{SCE/ cellule}

a été de 8, 68 t _{3, 24 pour} PKW et

_{6, 96 ! 3,45}

pour PHA et la

_différence

a été

_{significative.}

La distribution de

fréquence

_pour l’ensemble des

mitogènes

s’est très bien

_ajustée

à un

_mélangé

de dezlx lois de

_{Poisson, correspondant}

aux deux

_populations

de cellules concernées _par

_{chaque mitogène.}

échange entre chromatides

/

phytohémagglutinine

/

phytolaque

/

comparaison

/

chèvres

INTRODUCTION

Sister chromatid

_exchange

_(SCE)

is considered to be an

_{important cytogenetic}

test

for

_monitoring

_cytogenetic

_damage

induced

_by

environmental

_mutagens

_(Carrano

et

_al,

₁₉₇₈₎

as well as

for

_detecting

chromosome

_instability

conditions such as the

Bloom

_syndrome

in humans

_(Chaganti

et

_al,

_1974).

SCE studies

_reported

in humans as well as in other mammalian

_species

have

largely

indicated several

_important

factors that can

influence

the

frequency

of

SCE: BUdR concentration

_(Wolff

and

_Perry,

_{1974; Kato,}

_1974),

visible

_light

(Ikushima

and

_Wolff,

_1974),

_type

and amount of serum

_(Kato

and

_Sandberg,

1977),

exogenous viruses

(Kato,

1977),

cell

cycle

duration

_(Snope

and

_Rary,

_1979),

growth

temperature

(Speit, 1980),

composition

and

_type

of medium

_(Mutchinick

et

_al,

_1980),

_proportion

of B and T

_lymphocytes

_(Lindblad

and

_Lambert,

_1981),

antibiotics and serum

_(Das

and

Sharma,

1983),

sex and _age

(Soper

et

_al,

_1984),

dietary

habits

_(Wulf

et

_al,

_{1986), group,}

animal and BUdR treatment

_(Catalan

et

_{al, 1994;}

Iannuzzi et

_al,

_1991a).

As is

_known,

in vitro SCE studies are

_routinely

carried out on

_peripheral

blood

_lymphocytes

stimulated either with

_{phytohemagglutinin}

_(PHA-M

_form)

or

with

_pokeweed

_(PKW)

_mitogens;

the choice between them

_{mainly depends}

upon how much

_{hemagglutination}

is tolerated in the

_{cultures; furthermore,}

_pokeweed

stimulates both classes of B and T

_lymphocytes,

whereas

_{phytohemagglutinin}

mainly

stimulates T

_lymphocytes

_(Rooney

and

_{Czepulkowsky,}

_1986).

Since the

proportion

of B and T

_lymphocytes

in the blood

_and,

to a

_{greater extent,}

the rate of cell

proliferation

in the culture

_system

have been indicated as

_important

factors

affecting

the

_SCE/cell

_frequency

_(Santesson

et

_{al, 1979;}

Lindblad and

_Lambert,

1981)

it is

_likely

that the

_mitogen

used in the culture

system

might

well influence the final

_{SCE/response.}

The

_{present study}

refers to the differential

_SCE/cell

_response observed in blood

lymphocytes

of

_goat

_{( Capra}

hircus

_L)

stimulated with PHA and

_PKW,

and

_exposed

to final concentrations of

_{0.1, 0.25,}

_{0.5, 1,}

2.5 and 5

_vg/mL

of BUdR.

MATERIALS AND METHODS

Venous blood was

aseptically

collected from four

_goats

(two

males and two

_females)

Aliquots

of 0.5 mL of whole

_heparinized

blood were cultured at 37.5 °C in 9.5 mL of RPMI 1640 medium

_(Gibco,

Dutch

_{modification),}

_containing

10%

fetal calf serum

(Gibco),

0.1 mL

_L-glutamine

_(Gibco),

30

_vL

of antibiotics and 50

_!iL

of

_fungizone.

For each animal 12 culture flasks were

prepared,

six stimulated with 0.1 mL

phytoemagglutinin

and six with 0.1 mL

_pokeweed

_mitogens

_(both

from

_Gibco).

After 36 h of

_growth,

BUdR

_(Sigma,

Saint

Louis, MO,

USA)

was added at final

concentrations of

_{0.1, 0.25, 0.5, 1,}

2.5 and 5

_pg/mL,

_{respectively,}

for the PHA

and PKW sets of flasks. The cultures were

_protected

from

_light

and allowed to

grow for an additional 36 h. Colcemid was added for the final 60 min. Harvested

cells were treated with

_hypotonic

solution

_(KCI,

0.075

_M)

for 20 min at 37.5 °C and fixed three 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 second

cycle metaphase

spreads, randomly

scored for each

_animal,

for each BUdR level. All

_scoring

was

_performed

_by

the same

person.

Statistical note: For each BUdR dose and for both

_mitogens,

data were

anal-ysed by

means of Poisson’s

_probability

_function,

where the

_expected

values were

calculated

_using

the

_following

formula:

The chi _square method was utilized to estimate the

_goodness

of fit between observed and

_expected

values. At the lowest BUdR dose Poisson’s

_probability

function fit very well the observed data for both PHA and PKW

mitogens.

Conversely,

at the

_highest

BUdR dose the fit was not observed for either

_{mitogens. Therefore,}

at

5.0

_!tg/mL

of BUdR a Poisson ’mixture’ model was used. The ’Poisson mixture’ is a

non-linear

_regression

function that allows the

_estimation,

_through

the least _squares

method,

of the unknown

parameters

y,

!1, !2,

which minimize the

expression

.!1

and

A

2

represent

the

_{’means’, -!}

and

_{(1 - ry)}

the relative

_percentages

of the two

subpopulations

of B and T

_lymphocytes.

The fitness of the function is evaluated

through

the

R

2

coefficients.

RESULTS

Table I shows the individual mean rates and standard deviations of

_SCE/cell

at

_increasing

doses of BUdR in PHA and PKW stimulated

_goat

_lymphocytes.

At 0.1

_vg/mL

of BUdR the

_SCE/cell

rates of the two

_mitogens

are _very similar

(3.28 !

1.76 for

PKW,

3.17 ! 1.93 for

_PHA),

the difference not

_{being statistically}

significant;

at 5.0

_!g/mL

of

_BUdR,

PKW values are

_{significantly higher}

_compared

to the PHA ones

(8.68 ! 3.24

versus

6.96 ! 3.45,

respectively) (P

=

0.0001).

From _a

BUdR concentration of 0.25

_ug/mL

and

_above,

PKW values are

_{significantly higher}

than PHA values.

increase more

_slowly

and

_{regularly compared}

to PKW ones, which increase more

rapidly

from 0.1 to 0.5

_!g/ml

_{of BUdR,}

remain

_fairly

constant from 0.5 to 1

_[i

_g/mL,

and increase

_again

_up to 5

_wg/mL.

_{By extrapolating}

the two curves

_beyond

the

dosage

of 5

_!Lg/mL,

the

_SCE/cell

values scored on PKW stimulated

lymphocytes

would

_likely

continue to remain

_higher

_compared

to those for PHA.

In order to

_verify

whether the number of cells examined would have _any

significant

effect on the estimated means we extended the observations to 50 more second

_{cycle metaphase}

_plates

at

_dosages

of 0.1 and 5.0

_vg/mL

of BUdR.

_By

doubling

the number of observations from 50 to 100 the ’means’ estimated over

the first 50 do not

_{change significantly.}

In order to

_study

the variation in the

_SCE/cell

distributions within the cell

pop-ulations, only

the lowest and the

_{highest dosages}

were selected and the individual

SCE/cell

values were scored on 100 cells for each donor and

_pooled,

_summing

_up

to 400 cells for each BUdR level.

Figure

2 shows the observed

_(obs),

Poisson

_expected

_(exp)

and Poisson ’mixture’

expected

(exp a)

SCE/cell

distributions at 0.1 and 5

_!g/mL

of

_{BUdR, respectively,}

for PHA and PKW stimulated

_lymphocytes.

At 0.1

_!ig/mL

of BUdR both

_mitogens

behave in a similar

_fashion,

and the Poisson

_frequencies

fit _very well

_(chi

_square = 15.31 for PHA and 6.98 for

_PKW;

chi

square 0.05 = 18.3 and

_15.5,

respectively).

At

mitogens

deviate

_{significantly}

in their

behaviour,

and the Poisson

frequencies

do not

fit _anymore

_(chi

_square = 49.3 for PHA and 28.61 for

PKW;

chi

square 0.05 = 25

and

_26.3,

_{respectively).}

_However,

when the SCE

_{expected frequencies}

are calculated

on the basis of a Poisson ’mixture’

function,

_{they again}

fit the observed

_frequencies

(chi square

= 5.4 and 13.56 for PHA and

PKW,

respectively: adjusted

R

2

= 0.98

and

0.94,

respectively).

DISCUSSION

The results of the

_present

_{study clearly}

indicate that at 0.1

_¡.¡.g/mL

of

_BUdR,

the

two

_mitogens

have

_SCE/cell

rates that are

_strikingly

similar to each

_other,

and the

Poisson model fit very well the observed

frequencies.

Since BUdR concentrations lower than 0.1

_I

_vg/mL

do not allow a clear differential

_staining

between the sister chromatids

_{(Kato, 1974),}

it is

_likely

that the

_exchanges

observed at this

_dosage

are not BUdR induced or

_they

are so

_only

to a minimum extent. Previous data

on the estimation of the

_spontaneous

rate of sister chromatid

_exchanges

in cattle

(Di

Berardino et

_al,

₁₉₉₅₎

and

_goat

_(Di

Berardino et

_al,

₁₉₉₆₎

have shown that 0.1 I

!Lg/mL

of BUdR can be considered _very close to the level of

spontaneous

SCEs. From 0.25

₄

_g/mL

_up to 5

₄

_g/mL

of

_{BUdR, pokeweed}

stimulated

_goat

lympho-cytes

exhibit

_SCE/cell

rates

_{significantly higher}

compared

to

_{phytohemagglutinin.}

As shown in

_figure

_1,

_by

_{extrapolating}

the two curves

_beyond

5

_!tg/mL

of

_BUdR,

_up

to 10 or even 20

_¡.¡.g/mL,

which

_represent

the

_{dosages mostly}

used for SCE

_studies,

the mean

SCE/cell

rates scored on PKW stimulated

lymphocytes

would continue

to remain

_{higher compared}

to those achieved on PHA. One of the most reasonable

explanations

for such a difference can be found on the different cell

_targets

of the

two

_{mitogens: PHA,}

in

_fact,

stimulates

_mainly

T

_cells,

while PKW stimulates both T and B cells. Extensive SCE data on B and T human

_lymphocytes

are

_{reported by}

Santesson et al

₍₁₉₇₉₎

and

_by

Lindblad and Lambert

₍₁₉₈₁₎

who found

_{significantly}

’higher’

SCE/cell

values correlated with ’lower’

_{proliferation}

rates in T

_compared

to B

_lymphocytes.

The authors stated that the

_major

determinant of the

_SCE/cell

frequency

may not

simply

be the

proportion

of B and T

lymphocytes

in the

periph-eral blood but the rate of cell

proliferation

in the culture

system;

this

conclusion,

however,

has been confuted

_by

other researchers who found no correlation between

SCE/cell

frequency

and cell

_cycle

_(Giulotto

et

_{al, 1990; Loveday}

et

_al,

_1990;

Steinel

et

_{al, 1990;}

Catalan et

_al,

_1994).

As

_{suggested by}

Kato and

_Sandberg

_(1977),

and

by

Lindblad and Lambert

_(1981),

such a

_{discrepancy might}

be accounted for

_by

the

presence of different

subpopulations

or

clones,

within the B and T

lymphocytes,

differing

in BUdR

_sensitivity

or other SCE

_inducing

factors,

which _may further

contribute to the enhancement of the

_{variability normally}

observed in the SCE results.

At 5

_qg/mL

of

_{BUdR, ie,}

under the conditions of BUdR-SCE

_induction,

the Poisson model does not fit the observed

_frequencies,

unless a ’mixture’ model is used. This

_finding

_may

_provide

an

_explanation

for

_{discrepancies}

with SCE data

furthermore,

the authors

reported

that the

_SCE/cell

_frequencies

did not follow the Poisson

_{distribution,}

as we also

_report

here.

_Probably,

even in that _case, a Poisson ’mixture’ would have fit

_properly,

if tested. The same considerations hold for the

SCE data

reported

in cattle and river buffalo

_by

Iannuzzi

et al

_(1988,

_1991a,b).

The

_present

data are also

_slightly

_higher

_compared

to those

_reported

_by

Sanchez and

_Burguete

₍₁₉₉₂₎

on the

spanish

Murciano-Granadina breed of

_goat,

but since

they only

examined one

_donor,

their

_study

can be considered

_only

as indicative. In

_conclusion,

the data

_presented

herein show

_that,

under BUdR

_dosages

nor-mally

utilized for ’in vitro’ SCE

_{studies, ie,}

from 5 to 20

_{vg/mL (final}

concen-tration): (a)

the

_SCE/cell

rates based on PHA stimulated

lymphocytes might

be underestimated

_compared

to PKW and _{vice versa;}

_(b)

the Poisson ’mixture’

prob-ability

function is more suitable than the

_simple

Poisson to describe the

_SCE/cell

distribution. These considerations _may be of some

_importance

when

_comparisons

are to be made between SCE data obtained in different laboratories

_using

one

mi-togen

or the other. In our

_{opinion, however,}

the

_SCE/cell

rates shown

_by

PKW should be considered more

_meaningful

than those obtained with

_{PHA, being}

rep-resentative of the entire

_population

of B and T

_lymphocytes

_{and, therefore,}

of the whole individual.

SCE

studies,

alone or in

_conjunction

with

chromatid/chromosome

aberrations and

_micronuclei,

are of

_great

_importance

in the domestic animal

_industry,

because

they

allow detection of

genotoxic

effects induced

_by

environmental

_mutagens

such as

_{mycotoxins, pesticides, heavy metals,}

and so _on, which _may affect not

_only

the animals but also the human

_workers,

_directly

or

through

possible

residues in food-stuffs of animal

origin

(Rubes,

1987).

Breeding

animals, expecially

those utilized in artificial

_{insemination,}

should be checked and also selected on the basis of the

SCE/cell

rate,

in order to eliminate the risk of

_spreading

_genetically

_unstable

geno-types

into the

population,

thus

_compromising

the

_{genetic improvement}

_programs. ACKNOWLEDGEMENT

We thank C Vitale of the

_Department

of

_{Statistics, University}

of

Salerno,

for his valuable collaboration in statistical

_analysis.

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