Sister chromatid exchange test detection of toxin-induced damage in cultured fish

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Sister chromatid exchange test detection of

toxin-induced damage in cultured fish

J Lobillo, Jv Delgado, A Rodero

To cite this version:

Sister chromatid

_exchange

test

detection

of toxin-induced

_damage

in cultured fish

J Lobillo JV

_Delgado

A

Rodero

Facultad de

I!eterinaria,

Universidad de

_C6rdoba,

DepartaTnento

de

Gen6tica,

Avda Medina

_{Azahara, 9, 14005 Cdrdoba, Spain}

(Proceedings

of the 9th

European Colloquium

on

_Cytogenetics

of Domestic

_Animals;

Toulouse-Auzeville,

10-13

_July

₁₉₉₀₎

sister chromatid _exchange

_{(SCE) /}

genotoxicity

/

fish

INTRODUCTION

In recent _years, industrial and

_agricultural

toxic wastes in waters from the

Guadalquivir

Basin,

Gulf of Cadiz and the Strait of Gibraltar have increased. This

growing

contamination has motivated our team to

_develop

the

_cytogenetic

sister chromatid

_exchange

_(SCE)

test,

with the aim of

_detecting

the effects of these wastes

on the autochthonous cultured and wild fish

_species

of the Southern Atlantic off

the coast of

_Spain.

The SCE test consists of the detection of increases in the normal mean number

of sister chromatid

_exchanges

in

_metaphase

M2 cells. These cells are those which

have

_undergone

two rounds of

_replication

in the presence of

5-bromodeoxyuridine

(BrdU).

The chromatic chromatid differentiation is

_easily

detected

_by

_simple

acridine _orange

_staining

or with the fluorescence

_plus

Giemsa

(FPG)

_technique.

MATERIALS AND METHODS

Fishes

_belonging

to Rutilus alburnoides

_(Teleosteens

_Cyprinidae)

and to Liza

aurata

_(Teleosteens

_Mugilidae)

species

were used in this

study

because of their

representativeness

of the Southern Atlantic

_{Spanish ichthyological}

fauna.

Four groups of individuals of these

_species

were maintained in well-aerated

aquaria

at 25-27°C.

_Forty-eight

hours before

sacrifice,

they

were

injected

ip

with

0.5 _mg of

_{phytohemagglutinin}

_(PHA-M)/

₅₀

_g of fish

_body

_weight.

One dose

_(0.8

mg

of

_BrdU/g

of

_fish)

was

_{injected directly}

into the

_cephalic

_kidney

_{24, 22, 20,}

16 or

12 h before

_sacrifice,

in order to obtain the maximum M2 cell

_(cells

with sister chromatid

_{differentiation)}

_yield.

_Finally

0.3 _ag of colchicine per gram of fish was

injected

ip

2 h before sacrifice.

After

sacrifice,

the

_kidney

was removed and macerated in 0.075 M KCl for

Air-dried

_preparations

were obtained and stained with acridine _orange

_(Dutrillaux

and

Lejeune,

1973).

RESULTS

The BrdU treatment time which offered the maximum

_yield

of M2 cells was 16 h for Rntilus alburnoides and 22 h for Liza aurata.

Figure

1 shows an M2

_metaphase

cell with 4 SCE. The

_image

_belongs

to the first

species

of

_triploid

nature.

Mean SCE values for 1VI2

_metaphase

cells were 3.57 ! 0.59 in Rutilus alburnoides and 2.91 ! 0.43 in Liza aurata. Table I summarizes the results of the SCE assay

DISCUSSION

Although

the

_meaning

and mechanism of SCE is still

unknown,

it has been demonstrated that most of the

_genetically

active chemical toxins

_{(mutagens and/or}

carcinogens)

induce

_significant

increases in SCE when

_they

are tested in in vivo animal

_systems

or in vitro

_systems

with activation

_(Stetka

and

_Wolff,

_1976;

Carrano

et

_al,

_1978;

_Latt,

_1982).

At

_present,

the SCE test is a _very sensitive and

rapid

method for

_detecting

chromosome

_mutagenicity

and

_provides

a

powerful

means of

detecting

environmental

_mutagens,

as a

_{complementary}

test to other tests of the same nature.

The

_application

of BrdU

_{incorporation}

to the detection of SCE in fish was used

for the first time

_by

_Klingerman

and Bloom

_(1976).

Since their

_report,

other authors have used the detection of SCE as a sensitive

cytogenetic

test for

_{determining chemically}

induced

_genetic

_damage,

in in vivo

(Alink

et

_{al, 1980;}

van der Hoeven et

_al,

_1982;

van der Kerkhoff and van der

_Gaag,

1985)

and in vitro

_(Barker

and

_Rackhan,

₁₉₇₉₎

_systems.

In this

_study

we established the mean number of SCE in fish maintained in

high

quality

water,

in the absence of toxins. This value constitutes the infrastructure of the SCE

_genotoxicity

_test,

because _any increase of this mean value in fish

_exposed

to

contaminated water indicates the _{presence in} these waters of substances

_producing

genetic

damage.

We are now

_testing

the

_{genotoxic activity}

of several

agricultural

pesticides

whose results will be

_presented

in the future.

REFERENCES

Alink

_GM,

Frederix-Wolters

_EMH,

van der

_Gaag

_MA,

van der Kerkhoff

JFJ,

Poels CLM

(1980)

Induction of sister chromatid

_exchanges

in fish

_exposed

to Rhine water. Mutat Res

78,

369-374

Barker

_EM,

Rackhan BD

₍₁₉₇₉₎

The induction of sister chromatid

_exchanges

in cultured fish cells

_{(A!aeca es!lenderes)}

_{by carcinogenic}

_mutagens. Mutat Res

_68,

381-387

Carrano

_{AV, Thompson LH,}

Lindl

_PA,

Minklre JL

₍₁₉₇₈₎

Sister chromatid

_exchanges

as an indicator of

_mutagenesis.

Nature _271, 551-553

Dutrillaux

B, Lejeune

J

₍₁₉₇₃₎

Coloration des chromosomes humains par 1’acridine orange

apr!s

traitement _par le

_{5-bromod6soxyuridine.}

CR Acad Sci Paris Ser D 3179-3180

Klingerman AD,

Bloom SE

₍₁₉₇₆₎

Sister chromatid differentiation and

_exchanges

in adult mudminnow

_{( U!rebra}

_lirni)

after in vivo _{exposure to 5-bromodeoxyuridine.} Chromosoma

56, 101-109

Latt SA

₍₁₉₈₂₎

Detection,

significance

and mechanism of sister chromatid

_exchange

formation. Past

_experiments,

current _concept, future

_challenges.

In: Sister Chromatid

Exciaange.

(Wolff

S,

ed)

John

_Wiley

&

Sons,

Inc, New York

Stetka

_DG,

Wolff S

₍₁₉₇₆₎

Sister chromatid

_exchange

as an _assay for

_{genetic damage}

in-duced

_{by mutagen-carcinogen.}

I. In vivo test for

_{compound requiring}

metabolic activation. Mutat

_{Res 41,}

333-342

van der Hoeven

_{JCM, Brugeman}

_IM, Alink

_GM,

Koeman JH

₍₁₉₈₂₎

The killifish