Induction of Micro Nuclei in Bone Marrow by Two Pesticides

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Ž .Mutation Research 439 1999 239–248

Induction of micronuclei in bone marrow by two pesticidesand their differentiation with CREST staining: an in vivo study

in mice

Rosadele Cicchetti ), Monica Bari, Gabriella Argentin

Department of Public Health and Cell Biology, UniÕersity of Rome ‘Tor Vergata’, Via di Tor Vergata 135, 00133 Rome, Italy

Received 6 August 1998; revised 3 December 1998; accepted 8 December 1998

Abstract

Ž . Ž .Two pesticides, organophosphate phosphamidon PHO and organochlorine dieldrin DED were assayed by the mouse

bone marrow micronucleus test, to ascertain whether they showed genotoxic activity in vivo. Two doses, sub-lethalŽ . Ž .PHOs3 mgrkg b.wt.; DED s60 mgrkg b.wt. and lethal PHO s5 mgrkg b.wt.; DED s90 mgrkg b.wt. , of each

substance were administered intraperitoneally to 9–10-week old CBA male mice, in acute and repeated exposure. The

sub-lethal dose was also administered at two different times and twice at 24-h intervals. Both PHO and DED proved able toŽ .induce a dose-dependent increase of micronucleated polychromatic erythrocytes PCE . The two pesticides also showed a

different detoxification time. Furthermore, the CREST staining with antikinetochore antibodies allowed us to conclude that

the two chemicals are clastogens. q1999 Elsevier Science B.V. All rights reserved.

Keywords: Mouse bone marrow micronucleus test; CREST staining; Pesticide; Phosphamidon; Dieldrin

1. Introduction

Although the pesticides are often very effective,

many of them represent a potential hazard and their

use worldwide gives rise to concern on health andw xenvironmental effects 1 . Concern arises in particu-

lar as to carcinogenic, neurologic, reproductive, im-munological and developmental effects that have

)

Corresponding author. Tel.: q39-6-72596052; Fax: q39-6-

72596053

w xbeen associated with pesticide use 2– 5 . Also, the

genotoxic effects of several chemical groups of pes-

ticides have been shown by in vivo and in vitrow xexperiments 6– 11 .

With regards to genotoxicity studies, special at-

tention has been focused on cytogenetic assays and

so chromosomal breakage and chromosome loss havebeen studied for a long time because they can cause

diseases that have been correlated also with cancerw xdevelopment. 12–14 .

The in vivo mouse bone marrow micronucleus

test allows an effective assessment of both chromo-

somal damage and chromosome loss induced by

1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .P I I : S 1 3 8 3 - 5 7 1 8 9 8 0 0 1 8 5 - 5



( ) R. Cicchetti et al.r Mutation Research 439 1999 239–248240

chemicals, because it is simpler and faster than

traditional chromosome analysis. Additionally, it

provides the advantage of taking metabolism into

account as the genotoxicity of a substance actually

results from the dynamic balance between its en-

zymic activation and its enzymic detoxification. Mi-

cronuclei can originate from acentric chromosome

fragments as well as from whole chromosome lag-

ging at anaphase of the division of the nucleated

precursor cells; they persist in cytoplasm for some

time and so may be scored at interphase in polychro-Ž . w xmatic erythrocytes PCE 15,16 .

An increase in the frequency of micronucleated

PCE is therefore an indication of aneuploidy or

clastogenicity induction. For this reason, micronuclei

have been widely used to detect chromosomal break-

age and chromosome lagging in vivo and in vitrow x17–19 .

Although the bone marrow micronucleus test isuseful to screen the chromosomal anomalies it does

not allow to discriminate between their induction by

aneugens and clastogens, following the conventional

procedure. An essential step in the development of

this test has been therefore to find many strategies to

that purpose, such as the measurement of the DNAw x w xcontent 20,21 , the C-banding detection 20,22 , the

w xfluorescence in situ hybridization 23 and the use of Ž .antikinetochore antibodies CREST staining . The

antikinetochore antibodies isolated from serum of

w xscleroderma pigmentosum patients 24 enables infact the discrimination between micronuclei contain-

Žing whole chromosomes positive to CREST stain-. Žing or chromosomal fragments negative to this

. w xstaining 25–29 . This approach is relatively fast

and simple and it has often been the method of

choice to establish the mechanism underlying mi-

cronuclei induction, also in the pesticide investiga-w xtions 30,31 .

Among pesticides, organophosphate and orga-

nochlorine are constantly a matter of discussion be-

cause of their wide use. The advantages of

organophosphates are their fast breakdown, with a

relatively fast disappearance of their residues from

plants, while the disadvantages are their considerable

and acute intoxicating effects. They have been found

to give rise to chromosome damage through acciden-w xtal and occupational exposure 32,33 . Organochlo-

rine pesticides are persistent, they are found as resid-

uals in the soil, in the body and in food and they

cause chronic toxicity.Ž .Phosphamidon PHO is an organophosphate pes-

ticide that causes teratogenicity and embryotoxicityw xin mice 34,35 and chromosome aberrations in man

w x w x33 and in mice, also inducing micronuclei 36,37 .Ž .Dieldrin DED is an organochlorinated pesticide

that has been found to be carcinogenic in mammalsw x w x38 , neurotoxic 39 and able to affect the immunity

w xsystem 40,41 . It is one of the most persistentw xcontaminants studied 42 , so much so that it was

w xfound at an application site 13 years later 43 .

Moreover, it is present in the human adipose tissue

and in maternal milk, even if no exposure is de-w xtectable 44–46 ; because of its liposolubility it is

w xalso found accumulated in food 47–50 .

In this study, PHO and DED were investigated by

the in vivo mouse bone marrow micronucleus assay

and the CREST staining method in order to assesstheir genotoxicity and to establish if they are aneu-

gens or clastogens.

2. Materials and methods

2.1. Animals and chemicals

CBA agouti male mice, 9–10 weeks old, wereused for all experiments. The animals were exposed

intraperitoneally to two pesticides widely used inŽagriculture, organophosphate PHO 0,0-dimethyl-0-

Ž .1-methyl-2-chloro-2-diethylcarbamyl vinyl phos-.phate, CAS No. 13171-21-6 and organochlorine

ŽDED 1-4,10,10 hexachloro-6,7-epoxy-1,4,4a,5-8,8a

octohydro-endo-exo-1,4,5,8-dimethanonaphthalene,.CAS No. 60-57-1 . The chemicals were diluted in

corn oil at concentrations chosen so as to allow thatŽ .the same vehicle amount 0.01 mlrg b.wt. was

always inoculated. Two different doses, sub-lethal

and lethal, were employed: the dose defined as

sub-lethal is the dose that allowed the sur ÕiÕal of

animals for at least 72 h and the lethal dose is the

one that allowed the sur ÕiÕal for at least 24 h. These

doses were determined through preliminary experi-

ments at 3 and 5 mgrkg b.wt. for PHO and at 60

and 90 mgrkg b.wt. for DED. Four or five mice



( ) R. Cicchetti et al.r Mutation Research 439 1999 239–248 241

Ž . Ž .Fig. 1. PCE with semi-filled upright triangle and without 'Ž .micronucleus and NCE without micronucleus ^ .

were used for each treatment group; the control

animals, two or three for each treatment, received

only an equal amount of vehicle.

2.2. Bone marrow micronucleus assay

The animals were sacrificed by cervical disloca-

tion 24 or 48 h after exposure according to theŽ .following experimental protocol: a sub-lethal dose,

Ž .24 h exposure time; b lethal dose, 24 h exposureŽ . Ž .time; c sub-lethal dose, 48 h exposure time; d

repeated exposure to the sub-lethal dose: animals

were treated twice with the lowest dose at 24-h

intervals and sacrificed 24 h after the last injection;Ž .e repeated exposure to the fractionated sub-lethal

dose: the lowest dose was subdivided into three

equal parts and was successively administered with a

gap of 24 h; the mice were sacrificed 24 h after theŽ .last exposure; f fractionated exposure to the lethal

dose: the same as previous procedure, but with the

highest dose.

After the established time for each experiment,

the mice were sacrificed, the femora extracted andthe bone marrow cells were collected in fetal calf

Ž .serum FCS ; then, the suspension was centrifuged

at 1000 rpm for 5 min and the pellet was carefully

resuspended in a little supernatant and used to per-

form the smears.

Table 1

Frequence of micronuclei in bone marrow PCE after treatment with two pesticides

Experiments Chemicals Dose Exposition Number MNr1000 PCE PCErNCE onŽ . Ž .mgrkg time h of mice from each animal 1000 erythrocytes

Ž . Ž .treated mean values"SD mean values"SD

Ž .a PHO 3 24 5 3.60"0.54)) 1.94"0.53

DED 60 24 5 2.40"1.14) 1.72"0.55Ž .b PHO 5 24 4 5.00"0.82)) 1.70"0.22

DED 90 24 5 4.20"0.84)) 1.86"0.27a bOIL CTRL 24 5 1.40"0.22 1.90"0.51

Ž .c PHO 3 48 4 3.25"0.96) 1.80"0.53

DED 60 48 4 3.00"0.82) 2.12"0.76aOIL CTRL 48 2 1.25"0.35 1.90"0.71

cŽ .d PHO 3q 3 48 5 2.40"1.34 1.78"0.54aOIL CTRL 48 2 1.50"0.00 2.00"0.85

Ž .e PHO 1=3 72 5 2.60"2.07 1.94"0.22

DED 20=3 72 5 2.00"0.71 2.50"0.59

Ž .f PHO 5r3=3 72 5 2.60"0.55) 2.02"0.66DED 30=3 72 4 1.75 q 0.96 2.50"0.90

a bOIL CTRL 72 5 1.90"0.65 2.10"0.65

)P-0.05 ))P-0.01.a Ž . b c

Same dose of oil used for all treatments 0.01 mlrg b.wt. ; Data on control mice were pooled; Data not available for DED because mice

died after the double injection.

MNs micronuclei; PCEs polychromatic erythrocytes; NCEs normochromatic erythrocytes; PHO s phosphamidon; DEDsdieldrin;

CTRLs control.




2.3. Slide preparation and staining

For the conventional assessment of micronucleus

frequencies, two slides for each animal were pre-w xpared according to the method of Schmid 51 .

For kinetochore identification, five slides for two

mice from experiments ‘a’ and ‘b’ were prepared

following the method described by Schriever-w xSchwemmer and Adler 52 . Briefly, the slides were

treated with 0.1% Triton X-100 in PBS for 3 min

and then incubated with antikinetochore antibodyŽ .solution CHEMICON for 45 min at 378C i n a

moist chamber. After two rinsings for 5 min in PBS

containing 0.1% Tween 20, fluorescein isothio-

Ž .Fig. 2. Distribution of mean frequencies of micronuclei induced by PHO and DED: comparison among different treatments. a Sub-lethalŽ . Ž . Ž .and lethal dose; b 24- and 48-h exposure times; c Single and double injection; d acute and repeated exposure to sub-lethal and lethal

dose. Sub-lethal dose: PHOs 3 mgrkg b.wt.; DEDs60 mgrkg b.wt. Lethal dose: PHO s5 mgrkg b.wt.; DEDs 90 mgrkg b.wt.

)P-0.05; ))P-0.01; SD are shown in Table 1.




Ž .cyanate FITC -conjugated goat antihuman IgG anti-Ž .body CHEMICON was added to the first antibody

and the slides were incubated for 45 min at 378C.

Finally, after two further rinsings as preÕiously de-

scribed , the slides were embedded in antifade solu-( )tion 2.5% DABCO, 90% glycerol in PBS contain-

ing 0.5 mgrml propidium iodide to counterstain

DNA.

2.4. Analysis

The conventional valuation of micronucleus fre-

quencies was assessed by light microscopy in at leastŽ1000 PCE per animal. The PCErNCE normo-

.chromatic erythrocytes ratio was also determined on

a total of 1000 erythrocytes counted.

The kinetochore identification was performed by

fluorescence microscope, scoring 100 micronuclei

per animal. Two bandpass filters were used: one at450–490 nm wavelength for simultaneous observa-

tion of fluorescein and propidium iodide and the

other with a wavelength of 510–560 nm for the

observation of propidium iodide fluorescence only.

The results were statistically evaluated by the

Mann–Whitney test, particularly indicated for small

samples and with possible wide variability intra

group.

3. Results

Genotoxic effects of PHO and DED were evalu-

ated by the detection of micronucleated cells, scoring

1000 PCE per animal in mice bone marrow smearsŽ .Fig. 1 .

Results of the induction of micronuclei in both

control and treated mice are shown in Table 1, where

the ratio between PCE and NCE on 1000 total

erythrocytes is also reported. This ratio is a useful

index to reveal the chemical toxicity affecting the

bone marrow cells: a significant decrease of

PCErNCE ratio in treated mice compared with

controls gives evidence of an erythropoiesis depres-

sion, with reduced proliferation of nucleated erythro-

cyte precursor cells. No significant reduction in the

PCErNCE ratio was found in all treated groups

compared to the control mice, so that no bone mar-

row toxicity was observed.

A significant increase of micronuclei with respect

to control mice was observed after treatment with

sub-lethal and lethal single doses at 24 h of exposureŽ .for both PHO and DED experiments ‘a’ and ‘b’ .

Likewise, a significant increase was also recorded inŽ .the 48-h exposure time experiment ‘c’ .

No significant difference in the micronucleus fre-

quencies was found in experiment ‘d ’, in which the

sub-lethal dose of PHO had been administered twice

with a gap of 24 h in between. This experiment was

also performed with DED, but the mice did not

survive the double injection of this substance.

Finally, fractioned dosing of both PHO and DEDŽcarried out with sub-lethal and lethal doses experi-

.ments ‘e’ and ‘ f ’ , induced a significant increase of

micronuclei only for PHO at lethal dose.

Fig. 2 shows the comparison among different

treatments of both chemicals.

Ž .A lethal dose of PHO and DED experiment ‘b’produced a significant increase of micronuclei if

Ž .these were compared Fig. 2a with those induced byŽ .the sub-lethal dose experiment ‘a’ .

The comparison between exposition to a singleŽ .sub-lethal dose for 48 experiment ‘c’ and 24 h

Ž .experiment ‘a’ did not show significant differencesŽ .Fig. 2b .

(The effect of double exposure to PHO experiment )‘d’ was significant lower than expected from the

sum of the effects at 24 and 48 h after single

( )exposure experiments ‘a’ and ‘ c’ .Lastly, repeated exposure of both chemicals in-

duced less effect than equivalent acute dosing, sig-

nificant for lethal dose alone.

Table 2 shows the data obtained by immunofluo-

rescent CREST staining to detect the kinetochores in

Table 2Ž q .Distribution of kinetochore positive KC micronuclei detected

with CREST serum

a qŽ .Chemicals Dose mgrkg MN scored KC MN

bOIL CTRL 200 0PHO 3 200 24

5 200 10

DED 60 200 17

90 200 0

aMN scored in two mice were pooled.

bSame dose of oil used for

Ž .the treatments 0.01 mlrg b.wt. .

PHOs phosphamidon; DEDsdieldrin; CTRL s control.




micronuclei. For each chemical, two animals were

treated with sub-lethal and lethal doses at 24 h and

100 micronuclei per animal were scored for theŽ q .presence of kinetochores KC MN . Colchicine

Fig. 3. Immunofluorescence staining with CREST antikinetochore antibodies and propidium iodide counterstaining for DNA. MicronucleiŽ .stained bright red, kinetochores bright yellow-green. a Mouse interphasic nuclei that contain fluorescent spots corresponding to

Ž . Ž . Ž y. Ž . Ž .centromeres; b and c PCE containing one micronucleus without kinetochore KC ; d the same field of c observed for DNA stainingŽ . Ž . Ž q. Ž .only; e and f PCE containing one CREST staining positive micronucleus KC ; in e the micronucleus shows three fluorescent spots,

Ž .in f one single spot. Wavelength for iodide propide: 510–560 nm. Wavelength for fluorescein and iodide propide: 450– 490 nm.




( )1 mgr kg b.wt., 24 h exposure time , was used as

positive control and gave KCq micronuclei with a( qfrequency of 73.5% 110 KC MN out of 150 MN

)scored . No discrimination between PCE and NCE

was needed for this analysis, since only one or two

micronucleated NCE were found in the fields con-Žtaining 1000 PCE in all experimental groups data

.not shown .

In Fig. 3, the yellow-green kinetochore spots

stained with CREST antibodies are identifiable in a

reddish background of propidium iodide as counter-

stain for DNA.

4. Discussion

The results clearly demonstrate that both PHO

and DED induce a statistical significant increase of

micronuclei either at sub-lethal or lethal dose; thisincrease was found to be dose-dependent. With re-

gards to PHO, our data confirm those obtained byw xUsha Rani et al. 36 at our maximum dosage and

w xby Behera and Bhunya 37 at the same doses as we

tested.

No comparable data are available in literature for

DED. Very limited studies on DED genotoxic effects

have been reported. Data on induction of micronuclei

and aberrations are however available for other

organochlorine compounds, such as aldrin and endo-

w xsulfan, for which Usha Rani et al. 36 did not reporta micronucleus increase at the tested dose; on the

w xcontrary, Georgian 53 observed a significant in-

crease of aberrations using high doses of aldrin inŽ .experiments in vivo 9.56 mgrkg b.wt. . For the first

time we have demonstrated the ability of DED to

induce genome damage.

No cytotoxicity was observed in both chemicals

and this is an important result because to be classi-

fied as genotoxic, a substance must be able to induce

genetic disorders at doses that do not damage the

organism with cytotoxic mechanisms.

It has been demonstrated that both chemicals are

able to induce a significant increase of micronuclei

with respect to those found in the control animals,

also when the mice were exposed at 48 h sub-lethal

treatment. When comparing the frequency of mi-

cronuclei found at different exposure times, we ob-

served, with the prolongation of the treatment time, a

slight decrease in the mice exposed to PHO and a

slight increase in the mice exposed to DED. Al-

though these differences did not attain statistical

significance, they might indicate that the two pesti-

cides have different toxicological properties.

In effect it has been reported that the detoxifica-

tion rate of organophosphorus compounds is very

fast in mammals and that these compounds are rapidlyw xmetabolized in vivo 54,55 , whereas the organochlo-

rine are considered to be among the most persistentw xcontaminants with a rather long half-life 42 .ŽThe double exposure to chemicals experiment

.‘d’ confirmed the different toxicokinetic properties

of the two pesticides. The DED-treated mice did not

in fact survive after the second injection, probably

because an interval of 24 h was not sufficient to

allow the detoxification of the compound and the

elimination of the metabolites. Therefore, the re-

peated dosing could have produced an increasedtoxic effect causing the animal’s death.

Ž .The PHO double treatment experiment ‘d’ in-

duced a micronucleus amount significantly lower

than the values expected on the basis of the results of

experiments ‘a’ and ‘c’. Actually, the effect of a

double injection should be close to the sum of the

effects produced by the single injections at the corre-w xsponding time 56 . So, if animals are injected at 0

and 24 h and tested at 48 h, the expected frequency

of micronuclei in the observed PCE should be the

sum of micronucleus frequencies at 24 h exposureŽ . Žexperiment ‘a’ plus that at 48 h exposure experi-

.ment ‘c’ . The result from the combined treatment

with PHO did not attain this expected value. Since

we can exclude, on the basis of the PCErNCE ratio,

a higher cytotoxicity of this double treatment, the

significant decrease of micronuclei in the double

treatment should remind the adaptive response that

enhances the resistance of the cells to a damaging

impact and gives additional capacities to repair. Thew xadaptive response, first reported in E. coli 57 , has

been found successively in various organisms ex-w xposed to clastogens, even in mice 22,58,59 .

However, the question as to why there is no

increase in micronucleated cells in the twice treated

animals still remains open for the time being, since

our data must be confirmed by suitable experiments,

particularly with regards to the first exposure dosing

usually very low in the adaptive response. It would




be very interesting to verify this hypothesis also for

DED, which has already been found to induce thew xadaptive response in the trout 60 .

The repeated exposure induced a significant in-

crease of micronuclei only in mice exposed to lethal

dose of PHO, with respect to control mice, and

always induced genotoxic effects lower than an

equivalent acute dose, with significant differences

for both lethal doses. This result could be due to a

daily lower intake which could enhance the effi-

ciency of the DNA repair mechanism, so that a

decrease in the micronucleus frequency was ob-

served.

Finally, we performed an immunofluorescentŽ .staining with antibodies antikinetochore Fig. 3 to

further investigate the mechanism underlying the

observed induction of micronuclei, since micronuclei

can result from chromosome breakage as well as

from chromosome malsegregation at mitoticanaphase.

A clastogenic mechanism was already taken into

account for both organochlorine and organophos-

phate pesticides because they induce chromosome

aberrations and because organophosphorus pesticidesw xare also known for their alkylating properties 61 .

The clastogenic effect was previously hypothesized

for PHO too by earlier studies that reported chromo-

some aberrations in human lymphocyte cultures andw xmouse bone marrow cells 37,53 .

Table 2 shows the distribution of micronucleiŽ q. qwith kinetochores KC . No KC micronuclei were

w xfound in the controls, according to Gudi et al. 62 ,

who in the in vivo mouse bone marrow cells did not

found spontaneous KCq micronuclei, using three

different solvents, whose one was corn oil. The fact

that the majority of PHO and DED-induced micronu-Ž y.clei were negative to the CREST staining KC

proves that the two pesticides are undoubtedly clas-

togens.

Inspite of the lethal dose induced a greater amount

of micronuclei than the sub-lethal one, the frequency

of KCq MN was lower at lethal dose. This result

could be explained, at least for PHO, admitting that

the higher dose produced an increase mainly of the

acentric fragments, causing the decrease in KCq MN

percentage.

In conclusion, our data confirm that the bone

marrow micronucleus assay, combined with the

CREST staining method, represents a useful tool to

demonstrate the genotoxicity of chemicals and their

action mechanism.

Acknowledgements

The authors thank Dr. Bruna Tedeschi for herinvaluable critical reading of the manuscript and Mr.

Carlo Idili and Mr. Graziano Bonelli for their techni-

cal assistance. This research was supported by a

MURST grant to Rosadele Cicchetti.

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Induction of Micro Nuclei in Bone Marrow by Two Pesticides

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