Amphibian micronucleus test(s): a simple and reliable method for evaluating in vivo genotoxic effects of freshwater pollutants
and radiations. Initial assessment *
Maria Fernandez, Jacques L'Haridon, Laury Gauthier and Catherine Zoll-Moreux Centre de Biologie du Developpement, UMR UPS / CNRS 9925 affilide ?t I'INSERM, Universitd Paul Sabatier, Toulouse, France
(Received 7 October 1992) (Revision received 16 March 1993)
A micronucleus test was developed using larvae from two urodele amphibians (Pleurodeles waltl and Ambystoma mexicanum) and an anuran (Xenopus laevis). The methods for maintenance of adults, egg laying, and rearing the larvae are described, and the conditions required for optimal response are given for each of these species.
The tests are carried out during a period of intense erythropoiesis when red blood cells are actively dividing in circulating blood. The micronuclei are observed on blood smears.
The genotoxic effects of X-rays were evaluated at 12 different doses over a range of 6-1200 rad. All doses, even the very low dose of 6 rad, gave positive results. The test substances were added to the water in which the larvae were reared, and the results obtained after treatment for 12 days a n d / o r 8 days with 47 different chemical compounds are listed. Detailed results are given as the lowest concentration producing a positive response or the highest concentration producing a negative response.
The reliability of the test system using the newt is now well established, while the tests using the other two amphibian species are still under evaluation. Integration of this test in a test battery for quality control of water would aid the evaluation of risks to human health, as well as the protection of aquatic ecosystems.
* In memory of Professor Andr6 Jaylet. Correspondence: Dr. M. Fernandez, Centre de Biologie du Developpement, UMR UPS/CNRS 9925 affili6e ~ FIN- SERM, Universit6 Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex, France. Tel. 33/61 55 69 52; Fax 33/61 55 65 07.
Living organisms are exposed to a wide variety of chemical compounds and various types of radi- ation, some of which may induce functional dis- turbances, reproductive dysfunction, or tumor formation as well as having genetic effects. In the 1920s, Muller (see De Marini et al., 1989) and
84
Mavor (see Cimino et al., 1986) demonstrated that X-rays produce gene and chromosomal mu- tations as well as aneuploidy. Over the ensuing decade various tumor-inducing chemical sub- stances were identified (see Osborne and Crosby, 1987).
The increasing realization that the environ- ment was becoming contaminated with chemicals, some being highly stable (e.g., organochlorine compounds), and the relationship established by Miller (1970) between modification of hereditary material and carcinogenesis, has stimulated much research on biological systems which can be em- ployed as bioassays for genotoxic agents. How- ever, in view of the multiplicity and complexity of processes involved in tumor formation, the devel- opment of tests for the detection of carcinogens is not straightforward, especially as some carcino- gens are not genotoxic. Nevertheless, the rela- tionship between qualitative a n d / o r quantitative alterations of the genome (gene, chromosomal or genomic mutations) and carcinogenesis is well established (Benedict et al., 1983; Kondo et al., 1984; Ramel, 1986). Although the results of short-term tests for mutagens may not be readily extrapolated to other species, they are of consid- erable value in risk evaluation.
In numerous eukaryotes, genomic mutations and certain chromosomal mutations can lead to the formation of micronuclei. In the animal king- dom, micronucleus tests using polychromatic ery- throcytes from bone marrow of rodents have been employed to evaluate the genotoxic potential of a wide variety of chemical compounds (Heddle et al., 1983; Natarajan and Obe, 1986). In aquatic vertebrates, this type of test can be used to assess the effects of contamination of the aquatic envi- ronment on the relevant species. However, detec- tion of genotoxic agents in water is of particular significance as aquatic pollutants may have harm- ful effects on non-aquatic species, including man, either via consumption of water (for review, see Meier, 1988) or via food chains.
To evaluate the overall impact of physical or chemical agents (or a combination of the two) on a whole organism, we have developed a micronu- cleus test in amphibians: the newt, Pleurodeles waltl, the axolotl, Ambystoma mexicanum, and the African toad, Xenopus laeL, is. The larvae of
these three species have nucleated red blood cells (RBCs) which divide actively in circulating blood (Deparis, 1973; Turner, 1988). The larval erythro- cytes thus represent a suitable cellular population in which cytogenetic damages from clastogenic and aneugenic agents can be readily detected. The micronuclei are counted on blood smears. We describe here the methodology and the exper- imental details pertaining to this test, and we present results obtained after exposure of the larvae to X-rays and various chemicals. The re- sults have been compared with those of other short-term genotoxicity tests (in vitro and in vivo) and carcinogenicity tests in rodents. The reliabil- ity of the amphibian micronucleus test, which has recently been validated (AFNOR, 1992), and its domains of application are discussed.
Materials and methods
Maintenance of adults The three species used: two urodeles, Pleu-
rodeles waltl and Ambystoma mexicanum, and the anuran Xenopus laevis adapt readily to perma- nent life in water, and so can be reared conve- niently in the laboratory. The adult males and females are reared in running water (dechlo- rinated tap water) in separate aquariums in groups of 10 individuals per 50-1 tank. The African toads (xenopus) are maintained as a temperature close to 18°C during the winter months. The animals are fed twice a week with meat (ox heart and liver) and chow (synthetic diet for trout; Aqualim, France).
Egg laying In the laboratory, the laying period (non-in-
duced) of the newt is from September to May with a slowing down between mid-December and mid-January. The female again attains reproduc- tive capacity 6 months after the last laying. Injec- tion of L H R H (luteinizing hormone releasing hormone) to the two partners can provide egg layings over extended periods of time (Grinfeld, unpublished results). Axolotl layings are comple- mentary to those of the newt and are available in summer and winter times.
For xenopus, egg laying can be induced throughout the year by injection of 300-750 IU
HCG (human chorionic gonadotropin) into the two partners. The selected couple is kept at 22- 24°C for 24 h before administration of hormone, which may be given in a single dose or in two doses on successive days. Eggs are laid in the following 12 h, and the female regains reproduc- tive capacity 3 months later. The pair are re- moved from the aquarium 24 h after egg laying.
Egg laying is abundant, as the two urodeles lay several hundred eggs at a time; the anuran lays several thousand eggs.
Embryonic and larval development
(a) Urodeles. After laying, the eggs are placed in bowls containing 5 -6 1 of tap water dechlori- nated by filtration through sand and active car- bon. The water temperature is maintained at around 18°C in an air-conditioned room. Since amphibians are poikilothermic, development can be speeded up or slowed down by altering the temperature. At 12°C, development is around 2-fold slower than at 18°C, whereas at 20°C it is 1.8-fold higher than at 18°C. It is thus easy to plan in advance any given stage of development.
However, the influence of temperature on cell division should be kept in mind, and at least 8 days stabilization at the test temperature should elapse before carrying out tests.
(b) Anuran. The eggs are laid in bowls in which the water is continuously aerated and kept at 22°C. Under these conditions hatching takes place 48 h after laying.
On the 6th day, the tadpoles are transferred to 50-1 aquariums (max. 500 individuals per tank). Homogeneous and rapid growth is obtained at a temperature of 22°C. At 18°C (a temperature at which development is around 2.5-fold slower), they must be stabilized at 22°C for 4 days prior to testing.
Feeding of larvae
(a) Urodeles. After hatching, the young lar- vae are fed ad libitum exclusively with live prey (larvae of Artemia salina or daphnia). They can then be placed in 50-1 aquariums (100 larvae per tank) containing a bed of sand with a closed-cir-
85
cuit water circulation. The animals may also be maintained in bowls (20 individuals per bowl) in which the water is changed daily. When they have reached a given size, they are maintained in run- ning water (dechlorinated) and fed on Chirono- mus larvae.
(b) Anuran. The vegetarian xenopus tadpoles are fed daily on a synthetic diet designed for ornamental fish. It is supplied as a powder in dehydrated form. The water should be renewed at least partially every 3 -4 days.
Treatment stage There are several requirements for selection of
the appropriate larval stage: (i) after the test, the larvae must be large enough to enable cardiac puncture and removal of enough blood for a smear; (ii) the test period must coincide with a period of intense hematopoiesis (corresponding to a period of active cell division in circulating blood; Deparis, 1973); (iii) the test must have ended before the onset of metamorphosis.
Morphological criteria for test larvae
(a) Pleurodele. The larvae must display an incipient fifth finger on the hind limb (stage 52b- 53 in the development table of Gallien and Durocher, 1957). At 20°C, this stage is attained around 8 weeks after egg laying. The first signs of metamorphosis appear 20 days later. At this stage, the animals are around 32 mm long, and grow a further 12 mm in the ensuing 12 days.
(b) Axolotl. The animals must display an in- cipient two first fingers on the hind limb, which is observed as a paddle with a central pleat. At 20°C, this stage is attained around 40 days after egg laying. The animals are 30 mm long at this stage, and grow a further 10 mm over the next 10 days.
(c) Xenopus. The animals must exhibit a con- striction at the base of the hind limb bud (stage 50 of the chronological table of Nieuwkoop and Faber, 1956). At 22°C, this stage is reached 15 days after egg laying. The animals are around 24
86
mm long at this stage, and double in size over the next 8 days.
Experimental conditions
(1) Exposure to chemicals
(a) Toxicity assay. For each substance tested, several groups of at least five larvae from the same laying were reared at 20°C (urodeles) or at 22°C (anuran) in water (100 ml / l a rva ) containing different concentrations of the substance under test. Control larvae were reared in water alone or containing the solvent (DMSO, ethanol, iso- propanol) that had been used to dissolve the test substances.
They were exposed to an alternation of attenu- ated natural light and darkness. The food (Chironomus larvae for the urodeles and syn- thetic diet for xenopus) and medium were re- newed daily.
This type of test lasted for 6 days and was used to determine the maximal concentration (MC) that could be employed in a genotoxicity test. The MC corresponds to half the concentration which does not induce any apparent sign of toxicity within 6 days.
(b) Genotoxicity test. This is carried out un- der the same conditions as described above, al- though each group contained more individuals taken from the same batch of eggs. The basal frequency of micronuclei varies from one laying to another and from season to season, and so a negative control must also be tested at the same time.
At the end of the t reatment period (8 or 12 days), a blood smear was taken from each animal. It was fixed in methanol (3 min), stained with Masson hemalun (10-12 min), rinsed in running water (10 min), and dried.
(2) Irradiation of laruae The X-ray source employed (80 kV, 3 mA)
produces a dose of 50 R / m i n to larvae placed 20 cm away from it. The pleurodele larvae at stage 53 were anesthetized by tricaine methanesul- fonate and then irradiated in groups of five just covered with water in a 5 cm diameter Petri dish.
Twelve groups of 30 animals were irradiated for different period of time, and the doses absorbed were measured with dosimeters supplied by the CEA (Centre d 'Energie Atomique). The animals were exposed to a single dose, and blood smears were taken on the 6 days after irradiation.
Chemicals The sources of the chemicals are specified in
the tables of results. Commercial carbaryl was purified according to Pipy et al. (1982) before use. Monochloramine, N-nitrosoatrazine, and N-nitrosocarbaryl were synthesized. Monochlo- ramine was prepared using a previously described method (Gauthier et al., 1989). The various stages of the synthesis of N-nitrosoatrazine were carried out in the dark; they have been described else- where (L 'Haridon et al., 1993). N-Nitrosocarbaryl was synthesized according to the method de- scribed by Elespuru et al. (1974) modified by Beraud et al. (1980).
Analysis of results The micronucleated cells and the number of
cell divisions were counted over 1000 erythrocytes on each blood smear. If the mitotic index of a group of treated animals was significantly below that of the control group, the results for this group were discarded.
The data were analyzed using the statistical method described by McGill et al. (1978). The confidence interval of the median at the 5% level is expressed by:
M +_ 1.57 x IQR/~/n
where M, IQR and n are median, interquartile range, and number of animals, respectively.
The difference between two medians M 1 and M 2 is significant if their two confidence intervals do not overlap (the result is considered positive).
Empirically, a result is taken as weakly positive when the median of the treated group is signifi- cantly different from that of the control group, but less than twice the control median.
Results and discussion
The amphibians of this study are widely used in research, especially in developmental biology.
They have long been used as test organisms in environmental toxicology. The embryos and lar- vae of these species have been found to be partic- ularly sensitive to aquatic pollutants (Greenhouse, 1976), and certain pollutants are known to be carcinogenic in these animals (Arffmann, 1964; J~inisch and Schmidt, 1980a,b). The two urodeles have large chromosomes (n = 12 in pleurodeles and n = 14 in axolotl) with a high DNA content (19.5 pg and 38 pg, respectively). Xenopus has chromosomes of smaller size (n = 18) and the DNA content is lower (3.1 pg) than that of the other two species.
Chemical compounds We evaluated the toxicity and genotoxicity of
chemicals currently employed in various areas of human activities (antioxidants, coloring agents, solvents, drugs, pesticides, etc.). Some of these compounds induce tumors by different processes and to various degrees in mammals, and possibly also in man. They include some alkylating agents (or direct mutagens such as ethyl methanesul- fonate, ethyl N-nitrosourea, aziridine, etc.), and agents requiring metabolic activation in order to interact with DNA (or promutagens such as benzo[a]pyrene, cyclophosphamide or nitrosodi- ethanolamine). Other compounds, although not intrinsically genotoxic (Aroclor, phenobarbital, 12-O-tetradecanoyl phorbol-13-acetate, etc.), may potentiate the effects of mutagens by epigenetic processes, via acceleration of metabolic transfor- mation of promutagens and/or disruption of the rhythm of division or disturbance of cellular com- munications.
The results obtained in larvae treated for 12 days are listed in Table 1, and those after 8 days treatment are listed in Table 2.
Out of 47 compounds tested in the pleurodele, 30 were tested for 12 days only, and nine com- pounds for 8 days only. Ten compounds were tested over 12 and 8 days, two of the results of which have not been presented in view of their toxicity. This is discussed below. In the axolotl, the effects of EMS and BaP were evaluated over 12 and 8 days, and various shorter periods of time. In xenopus, six compounds were tested, among which BaP is listed in Tables 1 and 2. In fact, this carcinogen was tested over 2-14 days.
87
When the compound was tested at various concentrations, the responses are shown for: the lowest concentration that led to a statistically significant increase in micronuclei (MN) or the highest concentration giving a negative response. When a compound was tested at a single concen- tration, this corresponded to the maximal concen- tration (MC).
The numbers in parentheses after the abbrevi- ation of a compound or a series of compounds are the reference numbers listed in the tables of results.
Cases in which the highest concentration does not correspond to the MC are indicated in paren- theses. The results obtained with BHA (6) at 2.5 ppm and mercuric chloride (23) at 24 ppb are listed for illustrative purposes. In both cases, the negative response was attributed to a toxic effect, either by blocking cell division (BHA) or by in- duction of anemia (HgCI2). For Aroclor (2) in the pleurodele, we show the results of two experi- ments carried out on the offspring of two pairs exposed to an identical concentration (0.05 ppm) at two different times of year. In spite of the difference in magnitude of the response, the neg- ative result in the autumn was confirmed in the spring (Table 1).
Out of the 38 compounds tested for 12 days in the pleurodele, three (sodium fluoride (NaF; 31), sodium nitrate (NaNO3; 33) and phorbol ester (TPA; 36)) were only tested at the MC. They all gave a negative response at this dose.
There is lack of agreement about the genotoxic effects of NaF (Li et al., 1988). These authors did not find any mutagenic action of NaF on Salmonella in the presence or absence of $9. Similar results have been reported by Ashby and Tennant (1991). In vitro, chromosomal aberra- tions have been found to depend on the type of cell (Ishidate et al., 1988). The negative result obtained in the pleurodele is in agreement with that observed in vivo in the mouse micronucleus test (see Li et al., 1988).
TPA is extremely toxic in the pleurodele. An equimolar concentration was found to be 10-fold more toxic than HgCl z and more than 100000 times more toxic than phenobarbital, another well-known promoter. It has been shown that TPA induces aneuploidy, chromosomal aberra-
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(EM
S)
65-5
0-0
62
31
+ 4.
7 25
.5
39
20
275
(2)
j
(2)
j
124
(2)
j
(C) X
enop
us la
evis
2 A
roel
or 1
254
d 32
5 0
1 +
0.4
0 1
17
(Aro
) 11
097-
69-1
0.
05
1 +
0.8
0 2
15
-
4 B
enzo
{a]p
yren
e d
252.
3 0
1 -t-
0.5
0.
5 2
20
(BaP
) 50
-32-
8 0.
06
4 +
1.2
3 6.
5 20
+
9 C
yclo
phos
pham
ide
mon
ohyd
rate
w
279.
1 0
2 +
0.7
1 3
19
(CP
) 60
65-1
9-2
5 3.
5 +
0.4
2.8
4 20
(+
)
12 D
imet
hyl
suif
oxid
e w
78
.1
0 2
+ 0.
5 1.
5 3
19
(DM
SO
) 67
-68-
5 55
0 1
+ 0.
5 0.
5 2
20
-
23 M
ercu
ric
chlo
ride
w
271.
5 0
3 -l-
0.8
2
4 17
(M
eC)
7487
-94-
7 0.
0012
5 7
+ 0.
8 7
9 17
+
25 M
ethy
l m
ercu
ric
w
chlo
ride
(M
MeC
) 25
1.1
0 3
+ 0.
8 2
4.3
20
115-
09-3
0.
0025
8
+ 1.
8 6
10.5
15
+
0.03
; 0.
12;
0.25
; 0.
5; (
1; 2
; 4)
(9)
e
(9)
j
2.5;
10;
20;
(9
) a
40;
80
275
(9)
j
0.00
25;
0.00
5;
(9)
d 0.
01
0.00
125;
0.0
05
(9)
d
Sol
vent
: w
, w
ater
; d,
DM
SO
_<
0.5%
o; e
, et
hano
l _<
0.2
5%r;
i,
isop
ropa
nol
(1.5
ppm
).
v +
, po
siti
ve;
(+),
wea
kly
posi
tive
; -,
ne
gati
ve.
* S
imil
ar n
egat
ive
resu
lts
wer
e al
so o
btai
ned
at p
H 6
and
pH
5;
* *
inhi
biti
on o
f ce
llul
ar d
ivis
ion.
C
once
ntra
tion
s in
par
enth
eses
gen
erat
e +
seve
re t
oxic
eff
ects
. *
(1)
Jayl
et e
t al
., 19
86a;
(2)
Jay
let
et a
i.,
1986
b; (
3) F
erna
ndez
and
Jay
let,
198
7; (
4) Z
oll
et a
l.,
1988
; (5
) F
erna
ndez
et
al.,
1989
; (6
) G
auth
ier
et a
l., 1
989;
(7)
Fer
nand
ez
and
L'H
arid
on,
1992
; (8
) L
'Har
idon
et
al.,
1993
; (9
) th
esis
res
ults
(F
erna
ndez
, 19
92;
Gau
thie
r, 1
989;
Gri
nfel
d, 1
983;
Zol
l-M
oreu
x, 1
991)
or
new
res
ults
. 4.
a:
Ald
rich
, S
tras
bour
g, F
ranc
e; b
: C
iba-
Gei
gy,
Rue
ii-M
alm
aiso
n, F
ranc
e; c
: F
luka
, B
uchs
, S
wit
zerl
and;
d:
Mer
ck,
Dar
mst
adt,
Ger
man
y; e
: M
onte
sano
, St
. L
ouis
, M
O,
US
A (
gift
); f
: P
ierc
e, R
ockf
ord,
US
A;
g; P
epro
, L
yon,
Fra
nce;
h:
Pro
labo
, B
orde
aux,
Fra
nce;
i:
Ser
va,
St.
Ger
mai
n-en
-Lay
e, F
ranc
e; j
: S
igm
a, S
t. L
ouis
, M
O,
US
A;
k: s
ynth
esiz
ed c
ompo
unds
.
TA
BL
E 2
FR
EQ
UE
NC
IES
O
F M
ICR
ON
UC
LE
AT
ED
R
BC
s (P
ER
10
00 C
EL
LS)
FO
LL
OW
ING
A
N 8
-DA
Y P
ER
IOD
O
F E
XP
OS
UR
E
Tes
t co
mpo
und
Mol
. C
onc.
M
icro
nucl
eus
freq
uenc
y N
um
ber
of
(Abb
revi
atio
n)
wei
ght
pp
m
Med
ian
+ C
on-
Low
er
Up
per
an
imal
s
CA
S n
um
ber
fi
denc
e in
terv
al
Qua
rtil
e Q
uart
ile
Res
ult
Oth
er c
onc.
te
sted
(pp
m)
Ref
. S
ourc
e
(A)
Ple
urod
eles
wal
tl
39 B
enz[
a]an
thra
cene
d
228.
3 0
4 _+
0.
8 4
6 15
(B
A)
56-5
5-3
0.18
75
7 +
1.4
6 9.
5 15
4 B
enzo
[a]p
yren
e d
252.
8 0
6 +
1.6
4 8
15
(BaP
) 50
-32-
8 0.
01
12
+ 2.
3 10
18
30
40 C
affe
ine
~ 19
4.2
0 5
_+
1.9
3 8.
5 20
(C
AF
) 58
-08-
2 10
0 34
+
4.3
25
40
30
41 C
apta
n d
300.
6 0
8.5
+ 1.
8 7
11
12
(CA
N)
133-
06-2
0.
25
13
+ 2.
2 11
16
13
42 C
arba
ryl
e 20
1.2
0 6
+_
1.2
3 6
15
(CA
RB
) 63
-25-
2 2.
5 21
.5+
4.
1 15
26
18
43 C
olch
icin
e w
39
9.4
0 5
_+
1.9
3 8.
5 20
(C
OL
) 64
-86-
8 15
6.
5_+
2.3
4 10
.5
20
11 D
ieth
yl s
ulfa
te a
15
4.2
0 7
_+
1.9
4 9
17
(DE
S) 6
4-67
-5
6 24
+
8 15
36
17
12 D
imet
hyl
sulf
oxid
e w
78
.1
0 7.
5_+
2.1
5 11
20
(D
MS
O)
67-6
8-5
1 10
0 7
_+
2.4
4 10
16
44 7
,12-
Dim
ethy
l-
benz
[a]a
nthr
acen
e d
256.
4 0
13
+ 1.
6 12
16
15
(D
MB
A)
57-9
7-6
0.01
25
43
_+
5.9
33
47
14
45 E
thyl
enei
min
e w
43
.1
0 3.
5_+
2.3
3 8
12
(ET
1) 1
51-5
6-4
0.5
59
+ 12
.3
42
76
20
(+)
+ + (+)
+ + + +
0.09
4; 0
.375
; 0.
75
0.01
25;
0.02
5; 0
.05
0.07
5; 0
.I;
0.25
; 0.
35;
0.5;
0.7
5
0.12
5
5 1.2;
12
137.
5; 2
75;
550
0.02
5; 0
.05
(7)
(9)
(9)
(9)
(9)
(9)
(1)
(9)
(7)
(9)
d i g C d J
16 E
thyl
met
hane
su
lfon
ate
" 12
4.2
(EM
S) 6
5-50
-0
18 N
-Eth
yl-N
'-ni
tro-
N
-nit
roso
guan
idin
e w
16
1.1
(EN
NG
) 42
45-7
7-6
46 F
orm
alde
hyde
w
30
(FA
) 20
6-44
-0
20 G
luta
thio
ne '~
30
7.3
(GSH
) 70
-18-
8
22 l
ndom
etha
cin
w
357.
8 (I
nM)
53-8
6-1
24 3
-Met
hylc
hol-
an
thre
ne a
26
8.4
(3-M
C)
56-4
9-5
47 N
-Nit
roso
carb
aryl
e
230.
2 (N
-CA
RB
) 70
90-2
5-7
(B) A
mby
stom
a m
exic
anum
4 B
enzo
[a]p
yren
e a
252.
8 (B
aP)
50-3
2-8
16 E
thyi
met
hane
su
lfon
ate
w
124.
2 (E
MS
) 65
-50-
0
(C) X
enop
us l
aevi
s
4 B
enzo
[a]p
yren
e a
(BaP
) 50
-32-
8
Foo
tnot
es:
see
Tab
le 1
.
0 6
+ 0.
7 5.
5 7
12
20
67
+ 15
.4
43.5
77
.5
12
0 5
+__
1.5
3 7
17
0.4
16
+ 3.
8 10
20
17
0 7.
5+
2.1
5 11
20
5
8 +
1.7
6 12
30
0 9
_+
2.2
6 11
.5
15
0.5
9 5:
2.
4 7
13
15
0 10
+
1.6
7 11
15
25
10
+
2.7
6 13
17
0 8
_+
2.8
5 12
15
0.
5 22
2 +
32.6
18
5 26
6 15
0 6
+ 1.
2 3
6 15
0.
125
23.5
+
2.6
18.5
27
.5
30
+ +
10;
50;
100
0.08
; 0.
8
10
0.25
; 0.
5; 1
0 5
+0
.9
0.02
5 9
+ 2.
8
0 7
+ 1.
1 24
15
+
2.7
3.5
6.5
5 11
6 14.5
8 18.5
20
20
18
19
(+)
+
0.05
; 0.
1; 0
.2;
(0.3
; 0.
5; 0
.7)
3; 6
; 12
; 37
; 62
; 12
4
252.
8 0
1 +
0.7
0.
05
4 +
1.1
1.5
5 13
17
0.25
; 0.
5
(9;
1)
(1)
(9)
(9)
(9)
(9)
(9)
(2)
(2)
(9)
94
tions, and polyploidy in human lymphocytes (0nfelt, 1986). Although in this test negative re- sults were obtained with Chinese hamster cells (Ishidate et al., 1988), the results are not readily comparable to our responses in the pleurodele as the doses used in vitro were 400- and 20 000-fold higher (for CHO-K1 and CHL cells, respectively) than the maximum concentration tolerated by newt larvae.
The MC of sodium nitrate was 8000 ppm (L'Haridon et ah, 1993), a concentration well above that found in natural water. This chemical has been shown to cause chromosomal aberra- tions in hamster lymphocytes in vitro (Ishidate et al., 1988), whereas in the rat in vivo, the results were found to depend on the duration of expo- sure (Luca et al., 1985).
The negative results observed for these three compounds require further confirmation.
Out of the 17 compounds tested in the pleu- rodele for 8 days, four were only tested at a single concentration: caffeine (CAF; 40), colchicine (COL; 43), ethyleneimine (ETI; 45) and formal- dehyde (FA; 46).
CAF and ETI led to significant formation of micronuclei (34 %00 and 59 %oo respectively). However, their mode of action (and hence the origin of the micronuclei) is probably different. Caffeine was not found to generate chromosomal aberrations in Chinese hamster cells, although it did potentiate the action of X-rays in these cells (Dulout and Natarajan, 1987). It has been re- ported, for example, that caffeine causes detach- ment of kinetochores and induces aneuploidy in mammals (Zinkowski et ah, 1989). The large size of the micronuclei induced by caffeine in the pleurodele suggests that they could derive from whole chromosomes. On the other hand, ETI proved to be a powerful clastogen towards newt larvae: the positive response recorded in the am- phibian micronucleus test with this chemical is in agreement with the results in the equivalent ro- dent system in vivo (see Natarajan and Obe, 1986).
In contrast, 5 ppm formaldehyde was not clas- togenic in the newt (see Table 2). This compound has not been found to increase the frequency of micronueleated polychromatic erythrocytes in bone marrow (Natarajan and Obe, 1986), al-
though it has been reported to be carcinogenic in the rat and mouse (INRS, 1982).
Colchicine (43) was used at a dose (15 ppm) which does not block cell division. It is known that this compound binds to heterodimers of tubulins, and can induce aneuploidy (for review, see 0nfelt, 1986). It gives a positive response in the mouse micronucleus test after intraperitoneal injection (Jensen and Ramel, 1980). Adler et al. (1991) showed that 71% of the micronuclei formed possessed a centromere (revealed by c- banding). A comparable result was obtained in vitro by Degrassi and Tanzarella (1988) in ham- ster cells (CI-1). Using anti-kinetochore antibod- ies, these authors estimated that 84% of the micronuclei possessed kinetochores.
The negative results obtained in the pleu- rodele were probably due to the higher resistance of the larvae to the contact action of this alkaloid. Tests after intraperitoneal injection of this com- pound should determine whether the mode of administration affects the response, as has been observed with adriamycin (Grinfeld, 1983).
A dose-response relationship was not ob- served for certain compounds. The level of mi- cronuclei did not change following a 2-4-fold increase in the concentration of carbaryl (42) or benz[a]anthracene (39) (Table 2). Other agents induced a positive response, but only at their MC. They included caprolactam (7), ethidium bromide (15) and pyrene (30): these results have been discussed elsewhere (Fernandez et al., 1989). This is also the case for sodium hypochlorite (32), monochloramine (employed to disinfect drinking water), and chloral hydrate (38) which is a known chlorination by-product of water (Meier, 1988). With respect to captan (41), it should be noted that this fungicide was tested at a concentration of 0.25 ppm over a period of 2-12 days, followed in some cases by a period of recovery in water. The toxicity of the compound after 12 days expo- sure was indicated by anemia and a level of micronuclei below that of the group exposed for 8 days. This result is not shown in Table 1, but it illustrates the difficulties involved in detection of weakly genotoxic agents (Fernandez and Zoll- Moreux, in preparation).
In these nine cases, micronucleus formation could have been secondary to disturbance of the
mitotic apparatus. It is known for example that 80-90% of the micronuclei induced by chloral hydrate possess kinetochores (Degrassi and Tan- zarella, 1988). Furthermore, numerous agents whose genotoxic effect does not increase with the concentration, act on spindles and can cause ane- uploidy (0nfelt , 1986).
Although relatively few agents have been tested in xenopus, the results obtained with this species can be usefully compared to those obtained in the pleurodele. Five compounds led to the formation of micronuclei in xenopus. EMS, BaP and CP reacted positively at 5-, 6-and 10-fold lower con- centrations respectively, in the newt than those that were effective in the African toad. In con- trast, the thresholds of the anuran to the well- known spindle poisons, mercuric chloride (1.25 ppb) and methyl mercuric chloride (2.5 ppb), were clearly lower than those in the newt (12 ppb for both compounds) (see Table 1). Thus, al- though xenopus appears to be more resistant to mutagens (direct or indirect), it seems to be more sensitive than the pleurodele to the action of aneugenic agents. Confirmation with other spin- dle poisons currently being tested (study in progress) would provide a further endorsement of the value of the xenopus micronucleus test.
It should be specified that the optimal dura- tion of the amphibian micronucleus test was de- termined with reference mutagenic substances. For all three species, a 12-day period of treat- ment was sufficient to obtain an optimal response when BaP and EMS were tested at their respec- tive MC and M C / 2 . However, this period of time does not give the greatest sensitivity in all cases. For example, when concentrations of M C / 1 0 were assayed on newt larvae, BaP and EMS yielded a maximum response after a 14- and 16-day exposure respectively, while DES did not significantly increase the basal level of micronu- clei (Jaylet et al., 1986a). On the other hand, the lowest efficient concentration of a given chemical may not necessarily be different at 8 and 12 days of treatment.
At any rate, positive responses may be ob- served on shorter periods of exposure (4 days). In the pleurodele, positive responses were observed after rearing larvae in the presence of BaP (0.25 ppm), EMS (50 ppm), E N N G (0.4 ppm) and
95
TABLE 3
FREQUENCIES OF MICRONUCLEATED RBCs (PER 1000 CELLS) IN PLEURODELES LARVAE FOLLOWING EXPOSURE TO A SINGLE IRRADIATION BY X-RAYS
Dose of Micronucleus frequency Number Result X-rays Median Lower Upper of (rad) +Confidence quartile quartile animals
Micronuclei were recorded on day 6 after the irradiation.
N-CARB (0.5 ppm). In contrast, CAN (0.25 ppm) and DES (12 ppm) were not clastogenic under these conditions (results not shown).
X-rays Only the larvae of pleurodele were exposed to
radiation. Frequencies of micronucleated ery- throcytes after a single dose of X-rays are shown in Table 3. The clastogenic effect of X-rays in- creased progressively from 6 rad up to 600 rad, the dose giving a maximal response. Toxicity in- duced by higher doses led to a fall in the number of micronuclei.
The mechanism(s) of action of X-rays have not yet been completely elucidated. This range of electromagnetic radiation is sufficiently energetic to induce radiolysis of water and produce oxy- genated free radicals, especially the hydroxyl rad- ical O H . and the superoxide radical anion 0 2 • These radicals are known to be particularly reac- tive towards biomolecules. X-rays may also ionize D N A directly (Ferradini and Pucheault, 1983). In any event, it is generally agreed that micronuclei induced by X-rays result from chromosome breaks. We observed this on spread chromo- somes.
9 6
T A B L E 4
S U M M A R Y O F R E S U L T S F O U N D W I T H Pleurodeles waltl
Chemical class Number of Positive Negative c h e m i c a l s r e s p o n s e s r e s p o n s e s
t e s t e d
M i s c e l l a n e o u s 2 4 9 15
A m i n e s ( a r o m a t i c ,
a l i p h a t i c ) 7 5 2
N i t r o s o c o m p o u n d s 5 5 0
P o l y c y c l i c
c a r b o c y c l e s 5 5 0
N - , S - , O - m u s t a r d s 2 2 0
A z i r i d i n e s 1 1 0
C a r b a m a t e s 1 1 0
O x y g e n a t e d s u l f u r 2 2 0
T o t a l 4 7 3 0 17
X - R a y s 1 2 d o s e s 1 2 0
The overall results obtained with the pleu- rodele are shown in Table 4. Positive results were obtained for X-rays and with 30 out of 47 tested compounds while 17 gave a negative response. The positive compounds included the five N- nitroso compounds (18, 19, 27, 28 and 47) and the five polycyclic aromatic hydrocarbons (4, 23, 30, 39 and 44).
For comparative purposes, we have collected literature data on other short-term genotoxicity tests and on long-term carcinogenicity assays in rodents (Table 5). We have calculated the per- cent concordance with the newt micronucleus test (the cases where divergent responses were ob- tained with a given compound were not taken into consideration). Paradoxically the test was
F o o t n o t e s t o t a b l e 5
+ , p o s i t i v e ; ( + ) , w e a k l y p o s i t i v e ; - , n e g a t i v e ; ? , i n c o n c l u s i v e ;
L . P , l i m i t e d p o s i t i v e ; L . N , l i m i t e d n e g a t i v e ; E . E , e q u i v o c a l
e v i d e n c e ; + / - , d i f f e r e n t r e s u l t s d e p e n d i n g o n t h e a u t h o r s
o r o n t h e c e l l t y p e s ; A , a n e u p l o i d y ; P , p o l y p l o i d y .
a F r o m U p t o n e t a l . ( 1 9 8 4 ) .
b N u m b e r s i n s q u a r e b r a c k e t s : p e r c e n t c o n c o r d a n c e w i t h n e w t
M N t e s t . R e f e r e n c e s : A d l e r e t a l . ( 1 9 9 1 ) ; D e M a r i n i e t a l .
( 1 9 8 6 ) ; H e d d l e e t a l . ( 1 9 8 3 ) ; I s h i d a t e e t a l . ( 1 9 8 8 ) ; J e n s e n
a n d R a m e l ( 1 9 8 0 ) ; K i e r e t a l . ( 1 9 8 6 ) ; N a t a r a j a n a n d O b e
( 1 9 8 6 ) ; M a v o u r n i n e t a l . ( 1 9 9 0 ) ; N e s n o w e t a l . ( 1 9 8 7 ) ; 0 n f e l t
( 1 9 8 6 ) .
T A B L E 5
C O M P A R I S O N O F Q U A L I T A T I V E R E S U L T S O B T A I N E D
I N N E W T W I T H V A R I O U S S H O R T - T E R M G E N O T O X I C I T Y
A N D L O N G - T E R M R O D E N T C A R C I N O G E N I C I T Y T E S T S
N e w t C a r c . in . A m e s C . A . R o d e n t s
found to agree better with the bacterial test (86%, 37/43) than with the rodent micronucleus test (71%, 22/31). In this latter, negative responses were recorded with ECH (13) and o-TOL (37), two known mutagens and carcinogens. On the other hand, Py (30) (classified as a co-carcinogen) and CAP (7) (non-carcinogenic in rodents) in- creased the level of micronuclei in the newt. Taken together these results show that amphib- ian larvae represent a useful model system for evaluation of the genotoxic potential of radiation or aquatic pollutants.
The newt micronucleus test can also be used to evaluate directly the genotoxic potential of surface water without requirement for concentra- tion of micropollutants (Gauthier et al., 1992). It thus represents a useful tool for those responsible for pollution control in rivers. At present, the impact of pollution of French rivers is evaluated in the newt and xenopus. This current study was designed to validate the xenopus micronucleus test.
The method is sufficiently sensitive to detect genotoxicity in the tap water supplied to the laboratory (Jaylet et al., 1987) and after chlorina- tion or ozonation (Jaylet et al., 1990). The test thus could be employed at various stages during the treatment of raw water for production of drinking water. We are currently collaborating with French water authorities on the use of this test.
Compared with in vitro tests, our biological test system takes into account the whole set of processes involved in genotoxicity. These include diffusion of the xenobiotic substance, bioaccumu- lation, metabolic activation, detoxification and differential sensitivity of tissues or organs. It can be employed to assess the real effect of pollutants in freshwater, as well as the impact of possible changes in their genotoxic and/or toxic potential- ities (Fernandez, 1992; Fernandez and L'Hari- don, 1992; Fernandez and L'Haridon, in prepara- tion).
At the present time, the sensitivity and reliabil- ity of the newt micronucleus test (Jaylet test) are established, and the test is now a French Stan- dard (AFNOR, 1992). More widespread use of this test for quality control of water, such as integration into a battery of tests, would help in
the evaluation of risks to human health, and in the protection of aquatic ecosystems.
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