58, 135–143 (2000)
Copyright © 2000 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Effect of Atrazine on Implantation and Early Pregnancy
in 4 Strains of Rats
A. M. Cummings, 1 B. E. Rhodes, 2 and R. L. Cooper
Reproductive Toxicology Division, MD-72, National Health and Environmental Effects Research Laboratory,
U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Received May 14, 2000; accepted July 24, 2000
Atrazine (ATR) is an herbicide that has been shown to have
adverse reproductive effects including alterations in levels of pituitary hormones such as prolactin (prl) and luteinizing hormone
(LH) in female LE rats when administered at doses of 200 mg/kg/
day for 1 and 3 days. Because the action of prl in promotion of
progesterone secretion is essential for the initiation of pregnancy
in rats, this study was designed to examine the effect of exposure
to ATR during early pregnancy on implantation and short-term
pregnancy maintenance. Rats were divided into two groups representing periods of dosing with ATR prior to the diurnal or
nocturnal surges of prl. Within each group, four groups consisting
of four strains of rats [Holtzman (HLZ); Sprague Dawley (SD);
Long Evans (LE); Fischer 344 (F344)] were each further subdivided into four ATR dosages. Rats were dosed by gavage with 0,
50, 100, or 200 mg/kg/day ATR on days 1– 8 of pregnancy (day 0 ⴝ
sperm ⴙ). All animals were necropsied on day 8 or 9 of pregnancy.
The 200 mg/kg dose of ATR reduced body weight gain in all but
one group. Two groups of animals dosed at 100 and 200 mg/kg/day
in the nocturnal dosing period showed an increase in percent
preimplantation loss, and both of these were F344 rats. HLZ rats
were the only strain to show a significant level of postimplantation
loss and a decrease in serum progesterone at 200 mg/kg/day both
following diurnal and nocturnal dosing. Doses of 100 mg/kg/day
also produced postimplantation loss following diurnal and nocturnal dosing, but progesterone levels were decreased only after
nocturnal dosing. Alterations in serum LH were seen in several
groups. Serum estradiol was significantly increased only in SD rats
dosed at the diurnal interval with 200 mg/kg ATR. We conclude
that F344 rats are most susceptible to preimplantation effects of
ATR and that HLZ rats appear most sensitive to the postimplantation effects of the chemical. LE and SD rats were least sensitive
to effects of ATR during very early pregnancy.
The research presented in this article was funded wholly by the U.S.
Environmental Protection Agency. It has been subjected to review by the
National Health and Environmental Effects Research Laboratory and approved
for publication. Approval does not signify that the contents reflect the views of
the agency, nor does mention of trade names or commercial products constitute
endorsement or recommendation for use.
1
To whom correspondence should be addressed. Fax: (919) 541-4017.
E-mail: [email protected].
2
Present address: Lineberger Comprehensive Cancer Center, University of
North Carolina, Chapel Hill, NC 27599-7295.
Key Words: atrazine; rat; early pregnancy; embryo implantation; strain difference; progesterone; prolactin; hormones.
The herbicide atrazine (ATR) is a member of a family of
chloro-s-triazines that are used in contemporary agricultural
applications and are frequently found in groundwater at low
ppb levels (Gressel et al., 1984; Sinclair and Lee, 1992;
Stevens and Sumner, 1991). Recent research has focused on
the potential reproductive effects of ATR in rats, including
mammary tumor formation in Sprague Dawley (SD) rats
(Stevens et al., 1994; Wetzel et al., 1994), alterations in hormonal profiles (Cooper et al., 2000; Simpkins et al., 1998), and
disruption of ovarian cycling (Cooper et al., 1996). As reported
by Cooper et al. (2000), the administration of ATR to ovariectomized rats suppressed the estrogen-induced surges of both
luteinizing hormone (LH) and prolactin (prl) in a dose-dependent manner in both Long Evans (LE) and SD females. A
decrease in LH and prl secretion after ATR exposure was also
reported by Simpkins et al. (1998).
Progesterone is essential for the initiation of pregnancy in
the rat and human (De Feo, 1967; Psychoyos, 1973). During
the first 10 days of pregnancy in the rat, there are two daily
surges of prl, a nocturnal surge with a peak between 0300 and
0500 h, and a diurnal surge peaking between 1700 and 1900 h
(Terkel, 1988). It is these surges of prl that are luteotropic,
leading to the rescue and maturation of the corpora lutea (CL)
and to the subsequent dramatic increase in the secretion of the
progesterone that, along with ovarian estrogen, directly permits
implantation of the embryo in the uterus (Morishige and Rothchild, 1974; Smith et al., 1976). In fact, previous work has
shown that the administration of bromocryptine, a dopamine
agonist that inhibits the synthesis and release of pituitary prl
(Barrow and Tindall, 1983), to rats during gestation day (GD)
1– 8 can block implantation, presumably due to its ability to
prevent prl secretion and thereby block the necessary rise in
serum progesterone (Cummings et al., 1991). In the human it
is LH that supports CL function prior to implantation and
human chorionic gonadotropin (hCG) that exerts postimplan-
135
136
CUMMINGS, RHODES, AND COOPER
tation support of the CL and progesterone secretion (Zeleznik
and Fairchild-Benyo, 1994).
Effects of ATR on pregnancy in rats have been investigated
to a limited extent. According to Infurna et al. (1988), the
administration of technical ATR to rats during GD 6 –15 had
no effect at the 70 mg/kg dose, whereas 700 mg/kg produced
maternal mortality. A report by Peters and Cook (1973) states
that 1000 or 2000 mg/kg ATR, administered on GD 3, 6, and
9 resulted in embryonic resorption and embryotoxicity. In
work reported by Narotsky et al. (1999), exposure of rats to
ATR during GD 6 –10 produced full litter resorptions at 200
mg/kg. None of these studies investigated the effects of relatively low doses of ATR or effects on implantation.
Our study is based on the potential for ATR to disrupt early
pregnancy and implantation in the rat via a suppression of prl
surges. The hypothesis was that if ATR suppresses prl during
early rat pregnancy, then such a suppression of either the
diurnal or nocturnal surge of prl by ATR would impair implantation in rats. The testing of this hypothesis has been done
indirectly: ATR was administered to rats during early pregnancy and the impact of the chemical on implantation and
pregnancy maintenance until day 9 was assessed. The two
different times of ATR exposure used correspond to intervals
preceding the diurnal and nocturnal prl surges (Terkel, 1988).
A previous investigation of strain sensitivity to ATR during the
postimplantation interval (Narotsky et al., 1999) led us to
incorporate four different rat strains. The strains employed
were 1) Holtzman (HLZ) rats, which are historically used in
physiologically based pregnancy studies (Yochim and De Feo,
1963); 2&3) LE and SD, strains that were previously shown to
have a sensitivity to ATR with respect to altered estrous
cyclicity (Cooper et al., 1995); and 4) Fischer 344 (F344),
animals that have been used in previous work on effects of
ATR on pregnancy (Narotsky et al., 1999). Thus, these studies
examine the possibility that different strains of rats are differentially sensitive to the effects of potential prl suppression by
ATR, leading to adverse effects on pregnancy.
MATERIALS AND METHODS
Animals. Four strains of rats were employed: HLZ (Harlan, Indianapolis);
LE (Charles River, Raleigh, NC; SD (Charles River, Raleigh, NC); and F344
(Charles River, Raleigh, NC). Animals were purchased at 70 days of age and
caged in pairs in a room maintained at 20 –24°C. Food and water were
provided ad libitum. The light:dark cycle was kept at 14:10 with the midpoint
of the cycle at 12 N. For experiments in which dosing was during the dark
phase, the light cycle was exactly reversed so that the dark portion of the cycle
was during the day, and a red light was used while dosing the animals. Rats
were permitted to acclimate for 1 week, after which daily vaginal smears were
initiated. When rats demonstrated two consecutive regular estrous cycles, they
were bred by caging with untreated males on the night of proestrus. Successful
mating was confirmed by the presence of sperm in the vaginal smear and the
finding of vaginal plugs in the pan under the mating cages. The day of the
sperm-positive vaginal smear, corresponding with the day of estrus, was
considered day 0. Dosing with ATR commenced on day 1. Because only rats
TABLE 1
Effect of Atrazine on Relevant Parameters of Early Pregnancy
Parameter
Treatment group
Diurnal dosing
HLZ
0 (vehicle)
50 mg/kg ATR
100 mg/kg ATR
200 mg/kg ATR
SD
0 (vehicle)
50 mg/kg ATR
100 mg/kg ATR
200 mg/kg ATR
LE
0 (vehicle)
50 mg/kg ATR
100 mg/kg ATR
200 mg/kg ATR
F344
0 (vehicle)
50 mg/kg ATR
100 mg/kg ATR
200 mg/kg ATR
Nocturnal dosing
HLZ
0 (vehicle)
50 mg/kg ATR
100 mg/kg ATR
200 mg/kg ATR
SD
0 (vehicle)
50 mg/kg ATR
100 mg/kg ATR
200 mg/kg ATR
LE
0 (vehicle)
50 mg/kg ATR
100 mg/kg ATR
200 mg/kg ATR
F344
0 (vehicle)
50 mg/kg ATR
100 mg/kg ATR
200 mg/kg ATR
Body weight
change a (g)
Uterine weight
(g)
Ovarian weight
(mg)
26.6 ⫾ 2.6
19.3 ⫾ 3.9
12.6 ⫾ 4.3
⫺17.9 ⫾ 10.6*
1.83 ⫾ 0.16
2.00 ⫾ 0.12
1.96 ⫾ 0.24
1.43 ⫾ 0.28
89.1 ⫾ 7.4
82.9 ⫾ 1.7
85.7 ⫾ 4.7
70.5 ⫾ 5.5*
23.2 ⫾ 2.1
17.5 ⫾ 2.5
12.0 ⫾ 1.9*
0 ⫾ 2.7*
1.77 ⫾ 0.80
1.64 ⫾ 0.13
1.86 ⫾ 0.10
1.84 ⫾ 0.10
78.3 ⫾ 2.5
73.8 ⫾ 1.7
72.6 ⫾ 3.7
77.7 ⫾ 2.2
16.6 ⫾ 2.3
17.4 ⫾ 2.9
12.7 ⫾ 2.9
5.0 ⫾ 2.7*
1.21 ⫾ 0.16
1.33 ⫾ 0.14
1.52 ⫾ 0.07
1.53 ⫾ 0.06
75.0 ⫾ 4.9
77.3 ⫾ 4.8
69.1 ⫾ 4.0
75.1 ⫾ 1.9
8.8 ⫾ 1.1
10.2 ⫾ 0.8
10.1 ⫾ 1.0
8.2 ⫾ 0.7
1.59 ⫾ 0.06
1.63 ⫾ 0.03
1.68 ⫾ 0.04
1.62 ⫾ 0.05
77.4 ⫾ 2.9
75.2 ⫾ 2.0
76.8 ⫾ 3.5
75.1 ⫾ 1.6
19.0 ⫾ 2.8
10.8 ⫾ 1.5*
1.5 ⫾ 1.9*
5.2 ⫾ 2.9*
1.56 ⫾ 0.17
1.84 ⫾ 0.15
1.54 ⫾ 0.14
1.54 ⫾ 0.18
96.7 ⫾ 6.8
83.6 ⫾ 2.9*
76.1 ⫾ 3.0*
80.8 ⫾ 4.1*
14.1 ⫾ 3.3
9.3 ⫾ 2.4
4.4 ⫾ 2.4*
6.6 ⫾ 2.3*
1.19 ⫾ 0.13
1.22 ⫾ 0.15
1.45 ⫾ 0.14
1.52 ⫾ 0.14
68.9 ⫾ 3.8
74.2 ⫾ 3.3
70.5 ⫾ 3.0
67.4 ⫾ 3.4
20.4 ⫾ 2.1
8.7 ⫾ 4.1*
7.8 ⫾ 2.1*
0.7 ⫾ 4.1*
1.07 ⫾ 0.10
0.93 ⫾ 0.10
1.25 ⫾ 0.10
1.24 ⫾ 0.15
73.9 ⫾ 2.9
70.3 ⫾ 4.3
71.3 ⫾ 3.2
71.1 ⫾ 3.0
9.4 ⫾ 2.5
9.6 ⫾ 2.4
2.4 ⫾ 1.1*
⫺4.2 ⫾ 2.0*
0.92 ⫾ 0.03
0.79 ⫾ 0.07
0.68 ⫾ 0.10*
0.57 ⫾ 0.09*
48.3 ⫾ 0.9
46.8 ⫾ 0.9
47.4 ⫾ 0.9
45.5 ⫾ 0.7
Note. Rats were dosed with ATR on days 1– 8 of pregnancy and killed on
day 9, at which time measures were taken. Data are expressed as means ⫾ SE.
ATR, atrazine; HLZ, Holtzman; SD, Sprague Dawley; LE, Long Evans; F344,
Fischer 344 rats.
a
Body weight change ⫽ (body weight on day 9) – (body weight on day 1).
*Data are statistically different from vehicle controls within each strain and
dosing interval at a significance level of p ⬍ 0.05.
with regular cycles are used and because both vaginal plugs and sperm in
smears are used to identify breeding, the historical rate of percent pregnant in
our laboratory is 90 –95%. Animals were randomized by assigning each to a
137
ATRAZINE AND IMPLANTATION
FIG. 1. Effect of atrazine on preimplantation Loss. The effect on preimplantation loss of four dose levels of ATR in four strains of rats following either diurnal
or nocturnal dosing was evaluated on day 8 or 9 of pregnancy after dosing during days 1– 8. Diurnal dosing ⫽ 1400 h. Nocturnal dosing ⫽ 0200 h. HLZ, Holtzman;
SD, Sprague Dawley; LE, Long Evans; F344, Fischer 344 rats. Data are expressed as percentages (⫾ SE) calculated as follows: {[(total # CL) – (total # implantation
sites)]/# CL} ⫻ 100. Data marked with an asterisk (*) are statistically different from vehicle-treated controls within each strain and dosing interval (p ⬍ 0.05).
treatment group at the time that they were identified as pregnant (day 0). Rats
were bred in a staggered fashion over time, and the estrous cycles were not
synchronized. Because the decision of when to breed a particular animal was
based only on her vaginal smear record, this protocol provided an effective
randomization process.
Experiment 1. Rats were dosed by gavage with ATR, following daily
weighing, on days 1– 8 of pregnancy at 1400 h. This corresponds to a period
prior to the diurnal prl surge. Technical ATR (6-chloro-N-ethyl-N⬘-(1-methylethyl)-1,3,5-triazine-2,4-diamine) was supplied by Novartis (Greensboro,
NC) and was of 97.1% purity. ATR was suspended in 1% methyl cellulose and
diluted so that animals received 2.5 ml/kg at each dose level. Groups of 8 –10
rats received 0 (1% methyl cellulose vehicle), 50, 100, or 200 mg ATR/kg/day.
On day 9, rats were decapitated at 1400 h and trunk blood collected. Following
separation from red cells, serum was frozen for later assay of LH, progesterone, and estradiol. Serum prl was not measured, as the experimental design
(including times and method of euthanasia) precluded the determination of prl
surge levels or patterns. At necropsy the following parameters were assessed:
body weight, number of implantation sites, number of corpora lutea (CL),
ovarian weight, uterine weight, and number of resorptions apparent by day 9.
Experiment 2. Groups of rats were treated and evaluated exactly as in
Experiment 1 except that ATR was administered at 0200 h (prior to the
nocturnal surge of prl) and rats were killed at 2100 h on day 8 of pregnancy (19
h after the last dose of ATR).
Radioimmunoassays (RIAs). Serum estradiol was measured by a double
antibody RIA kit obtained from Diagnostic Products (Los Angeles, CA), and
serum progesterone was measured using a coat-a-count RIA kit supplied by the
same company. The determination of LH was performed using materials
supplied by the National Institute of Arthritis, Diabetes, Digestive, and Kidney
Diseases. The iodination preparation was I-6, the reference preparation was
RP-2, and the antisera was S-8. Radiolabeling of the iodination preparations
was performed with 125I (New England Nuclear, Boston) using the Chloramine-T method of Greenwood et al. (1963), after which labeled hormone was
purified on a BioGel P-60 (Bio-Rad Laboratories, Melville, NY) column. The
assay for LH had a sensitivity of 15 pg/tube, and inter-and intra-assay coefficients of variation of 7.7% and 5.9%, respectively.
Statistics. Parametric data were analyzed by a 3-way ANOVA (dose ⫻
strain ⫻ time) using the General Linear Models (GLM) procedure of SAS
statistical software (SAS Institute, 1985). When the overall ANOVA was
significant at p ⬍ 0.05, comparisons between groups were made using t-tests
(Least Squares Means; SAS). In most cases, only comparisons across dose
within strain and time were made. For hormone levels in control groups,
comparisons across strain and time were made. Percent data (pre- and postimplantation loss) were analyzed by 3-way ANOVA as above after arcsin
square-root transformation. Significant differences between data points were
indicated when the p value was less than 0.05.
RESULTS
Exposure of rats to 200 mg/kg ATR produced a significant
decrease in body weight change in all groups except F344
(diurnal dosing) (Table 1). Dosing with ATR during the noc-
138
CUMMINGS, RHODES, AND COOPER
FIG. 2. Effect of atrazine on postimplantation loss. Diurnal dosing ⫽ 1400 h. Nocturnal dosing ⫽ 0200 h. HLZ, Holtzman; SD, Sprague Dawley; LE, Long
Evans; F344, Fischer 344 rats. Data are expressed as percent preimplantation loss (⫾ SE) ⫽ [(# resorptions)/(total # implantations)] ⫻ 100. Data marked with
an asterisk (*) are statistically different from vehicle controls within each strain and dosing interval (p ⬍ 0.05).
turnal interval resulted in significant effects on body weight
change at lower doses than were effective when given during
the diurnal interval. Despite the use of four strains of rats, two
times of dosing, and four dosage levels of ATR, a significant
increase in preimplantation loss was seen in only two groups:
F344 rats following nocturnal dosing with 100 or 200 mg/kg
ATR (Fig. 1). In one data set (LE rats, diurnal dosing), ATR
treatment appeared to reduce preimplantation loss. HLZ rats
alone showed a significant increase in postimplantation loss, at
both dosing intervals at 100 and 200 mg/kg ATR (Fig. 2).
Table 2 shows the percent pregnant which represents the level
of success of breeding in these strains. The table also shows the
rarity of Full Litter Resorption (FLR). When the number of
implantations per dam was evaluated, treatment groups showing a significant difference from control corresponded to
groups that also showed significant or a trend toward significant increases in the percent of preimplantation loss (Table 2;
Fig. 1). HLZ and F344 rats were also the only groups in which
a significant change in organ weights was seen. Ovarian weight
declined in all ATR-treated HLZ rats after the nocturnal dosing
interval and in those treated with 200 mg/kg ATR at the diurnal
interval (Table 1). Uterine weight was decreased in F344 rats
treated in the nocturnal interval with 100 or 200 mg/kg ATR in
parallel with significant changes in preimplantation loss (Table
1). In no case was there a significant change in the number of
CL (data not shown).
When progesterone was measured on day 9 of pregnancy,
HLZ rats alone showed a decrease in serum progesterone at
100 and/or 200 mg/kg ATR in both intervals of dosing (Fig. 3).
When SD rats were dosed at the 1400 h interval (diurnal) with
200 mg/kg ATR, a significant increase in serum estradiol was
seen; in no other treatment groups did this phenomenon appear
(Fig. 4). Estradiol was, in fact, undetectable in all F344 rats
dosed and killed in the afternoon (Fig. 4). A significant reduction in baseline serum LH was seen as a consequence of ATR
exposure in several cases: 1) HLZ/diurnal dosing/100 and 200
mg/kg ATR; 2) LE/ diurnal dosing/100 mg/kg; 3) LE/ nocturnal and diurnal dosing/ 200 mg/kg; and 4) F344/ nocturnal
dosing/200 mg/kg (Fig. 5). SD rats exhibited no effect of ATR
on LH levels (Fig. 5).
DISCUSSION
The action of ATR to produce a reduction in the amount of
weight gained during the first 9 days of pregnancy is an
indication of the general toxicity of the chemical and is not a
reflection of any change in uterine weight. Such a general
toxicity may have contributed to maternal toxicity and the
ATRAZINE AND IMPLANTATION
TABLE 2
Effect of Atrazine on Parameters of Early Pregnancy
Parameter
Treatment group
Diurnal dosing
HLZ
0 (vehicle)
50 mg/kg ATR
100 mg/kg ATR
200 mg/kg ATR
SD
0 (vehicle)
50 mg/kg ATR
100 mg/kg ATR
200 mg/kg ATR
LE
0 (vehicle)
50 mg/kg ATR
100 mg/kg ATR
200 mg/kg ATR
F344
0 (vehicle)
50 mg/kg ATR
100 mg/kg ATR
200 mg/kg ATR
Nocturnal dosing
HLZ
0 (vehicle)
50 mg/kg ATR
100 mg/kg ATR
200 mg/kg ATR
SD
0 (vehicle)
50 mg/kg ATR
100 mg/kg ATR
200 mg/kg ATR
LE
0 (vehicle)
50 mg/kg ATR
100 mg/kg ATR
200 mg/kg ATR
F344
0 (vehicle)
50 mg/kg ATR
100 mg/kg ATR
200 mg/kg ATR
n
%
Pregnant a
%
FLR a
No. Implantations/
dam b
8
7
8
7
100
100
100
100
0
0
0
25
14.3 ⫾ 1.2
14.3 ⫾ 0.4
13.3 ⫾ 1.7
12.1 ⫾ 1.8
12
11
11
10
100
100
100
100
0
0
0
0
16.2 ⫾ 0.3
15.3 ⫾ 0.9
14.3 ⫾ 0.7*
15.4 ⫾ 0.5
10
10
10
10
80
90
100
100
0
0
0
0
12.3 ⫾ 2.1
13.0 ⫾ 1.6
14.4 ⫾ 0.6
16.2 ⫾ 0.7
10
10
10
10
100
100
100
100
0
0
0
0
16.1 ⫾ 1.0
17.5 ⫾ 0.8
18.1 ⫾ 0.9
17.5 ⫾ 0.9
7
10
11
10
100
100
100
90
0
0
0
0
15.7 ⫾ 0.6
16.3 ⫾ 0.7
13.9 ⫾ 0.9
12.1 ⫾ 1.7*
10
10
10
10
90
90
100
100
0
0
0
0
13.2 ⫾ 1.7
13.0 ⫾ 1.8
15.9 ⫾ 0.5
14.3 ⫾ 1.1
10
9
10
10
90
77.8
100
90
0
0
0
0
14.4 ⫾ 1.7
11.4 ⫾ 2.2
15.5 ⫾ 0.6
14.5 ⫾ 1.7
10
10
10
10
100
90
60
70
0
0
0
0
11.8 ⫾ 0.4
10.0 ⫾ 1.2
7.1 ⫾ 1.9*
8.1 ⫾ 1.8
Note. Rats were dosed with ATR on days 1– 8 of pregnancy and
killed on day 9, at which time measures were taken. ATR, atrazine;
HLZ, Holtzman; SD, Sprague Dawley; LE, Long Evans; F344, Fischer
344 rats.
a
Data are expressed as mean percentage. % Pregnant ⫽ (number of dams
with implantations and/or resorptions)/(number of rats sperm ⫹) ⫻ 100. %
FLR ⫽ % full litter resorptions ⫽ (number of dams with all implantation sites
resorbed)/(total number of dams with implantation sites and/or resorptions)
⫻ 100.
b
Data are expressed as mean ⫾ SE.
*Data are statistically different from vehicle controls within each strain and
dosing interval at a significance level of p ⬍ 0.05.
139
pregnancy loss observed, but the effects of ATR on body
weight change are not consistently paralleled by effects on
pregnancy. F344 rats appear most sensitive to the preimplantation effects of ATR, and those effects occur only following
nocturnal dosing. The increase in preimplantation loss in F344
rats treated in the nocturnal interval with 100 or 200 mg/kg
ATR is paralleled by a decrease in uterine weight in those
animals alone. HLZ rats showed a trend toward increased
preimplantation loss following both dosing intervals. SD and
LE rats appear insensitive to effects of ATR on implantation.
The apparent high preimplantation loss in control LE rats
following diurnal dosing is the result of finding two animals in
that group that were not pregnant (zero implantation sites), a
situation which is most likely due to failure of the breeding
process. The finding of statistically significant preimplantation
loss in the F344 group suggests that ATR was successful in
reducing prl levels sufficiently to interfere with implantation in
that strain following nocturnal dosing. Further work to pursue
that possibility is needed. Percent postimplantation loss is an
indicator of the degree to which pregnancy maintenance is
compromised between implantation (starting on day 4) and day
9 of pregnancy. Such an effect, observed in HLZ rats, suggests
that the hormonal mechanisms regulating pregnancy maintenance in rats, including prl, LH, and decidual luteotropin
(Morishige and Rothchild, 1974) may be most sensitive to
disruption in this strain. In addition, in HLZ rats dosed with
ATR, there are parallel effects on preimplantation loss, postimplantation loss, and the level of serum progesterone, suggesting
that the decrease in serum progesterone may mediate the decrease in implantations and increase in resorptions. An additional observation in HLZ rats is the finding of decreased
ovarian weight, a phenomenon that may be related to CL
failure and decreased progesterone secretion.
In a related set of studies, Narotsky et al. (1999) examined
strain differences in the sensitivity of rats to ATR and bromodichloromethane (BDCM), a by-product of drinking water
disinfection. In those experiments, rats were dosed on GD
6 –10, a postimplantation, LH-dependent interval, and evaluated following parturition. When the pregnancies of ATR- or
BDCM-treated dams were allowed to proceed to term, an
all-or-nothing phenomenon of full litter resorptions (FLR) was
observed. Whereas BDCM treatment caused FLR in F344 but
not SD rats, ATR treatment (200 mg/kg/day) led to comparable
incidences of FLR in F344, SD, and LE rats (Narotsky et al.,
1999). Based on the current data, it is not known whether
further evidence of postimplantation loss and/or FLR might
have been observed if the pregnancies in which the dams were
treated on days 1– 8 were allowed to proceed to term. However,
such a finding would corroborate data reported by Narotsky et
al. (1999). The increase in preimplantation loss observed here
is likely the result of preimplantation exposure to ATR producing a suppression of prl levels, which in turn would impair
CL maturation, elevation of progesterone secretion, and the
140
CUMMINGS, RHODES, AND COOPER
FIG. 3. Effect of atrazine on serum progesterone. Diurnal dosing ⫽ 1400 h. Nocturnal dosing ⫽ 0200 h. HLZ, Holtzman; SD, Sprague Dawley; LE, Long
Evans; F344, Fischer 344 rats. Data are expressed as means ⫾ SE. Data marked with an asterisk (*) are statistically different from vehicle-treated controls within
each strain and dosing interval with a cutoff of p ⬍ 0.05.
promotion of implantation. Such a prl suppression, induced by
bromocryptine, has been shown to block implantation in a
similar model of early pregnancy exposure in the rat (Cummings et al., 1991).
The finding of no effect of ATR on serum progesterone in
three out of four strains of rats is surprising in light of the
essential role of prl in CL development and progesterone
secretion and also in light of the putative action of ATR to alter
serum prl (Cooper et al., 2000). The measurement of baseline
prl at necropsy on day 9 (with euthanasia performed rapidly
enough to prevent stress-induced prl elevation) was not considered useful, as it is the twice-daily surges of the hormone
that are important for the stimulation of progesterone secretion
(Smith et al., 1976). In a study such as that reported here, the
introduction of an additional variable, that of using a series of
sequential necropsies to determine the rise and fall of each prl
surge, for each time of dosing, dose level, and strain data point
was considered to be unjustifiable with respect to the number
of animals required relative to the amount of information
obtained. Although it is impossible to state with certainty the
effect of ATR on prl during early pregnancy in this study, the
data on progesterone suggest either that ATR, at these dosages,
had little or no effect on the surges of prl during early preg-
nancy, or that ATR-induced changes in prl did not affect CL
function sufficiently to interfere with pregnancy.
With the exception of one case (F344/diurnal dosing/all
dosages), treatment of every strain at both time intervals
yielded at least a trend toward reduced baseline serum LH, and
this effect was shown to be significant in six cases. This is not
a measure of LH surge suppression, but merely an assessment
of changes in baseline levels. In two of the six cases of baseline
LH suppression, there was also a significant increase in percent
postimplantation loss (HLZ/diurnal dosing/100 and 200 mg/
kg). In the other four cases, there is usually at least a trend
toward increased postimplantation loss that parallels the decline in LH in each group. The number of resorptions detectable on day 9 does not always reflect the full complement of
resorptions that might be seen later in pregnancy or near term.
In work reported by Narotsky et al. (1999), for example, FLR
was detected in rats at term following treatment with ATR
during early pregnancy.
Nocturnal but not diurnal dosing with ATR was effective in
increasing preimplantation loss in F344 rats. No time-of-dosing effects were observed in other strains for this end point.
Both nocturnal and diurnal dosing (independently) produced
postimplantation loss in HLZ rats, but no effects on this end
ATRAZINE AND IMPLANTATION
141
FIG. 4. Effect of atrazine on serum estradiol. Diurnal dosing ⫽ 1400 h. Nocturnal dosing ⫽ 0200 h. HLZ, Holtzman; SD, Sprague Dawley; LE, Long Evans;
F344, Fischer 344 rats. Data are expressed as means ⫾ SE. Data marked with an asterisk (*) are statistically different from vehicle-treated controls within each
strain and dosing interval with a cutoff of p ⬍ 0.05.
point were evident in other strains. Serum LH showed a mixed
pattern with respect to effects of dosing interval. In LE rats,
dosing at each interval reduced serum LH. However, in HLZ
and F344 rats only diurnal or nocturnal (respectively) dosing
was effective. The finding of increased serum estradiol was
seen in only one case, following diurnal dosing. The apparent
elevation of estradiol levels in this one group of SD rats may
be simply the result of variability in the data for that hormone.
Overall, the effect of different intervals of dosing depended on
the end point measured and the strain of rat employed.
A comparison of serum levels of progesterone, estradiol, and
LH on day 9 in control animals across strains yields some
interesting observations. In animals killed at 1400 h on day 9
(following diurnal dosing), serum progesterone levels were
significantly higher in F344 and HLZ animals than in SD or LE
rats. At the same necropsy time, estradiol levels in F344 rats
were lower (undetectable) than those of the other three strains,
and serum LH showed a pattern where F344 and HLZ rats were
lowest, SD rats were intermediate, and LE rats were significantly higher than all others. In animals killed at 2100 h on day
8 (following nocturnal dosing), serum progesterone levels were
again high in the F344 strain and low in the SD strain, but in
LE rats progesterone was high whereas HLZ rats showed low
levels of the hormone. At 2100 hr, estradiol levels were much
higher in LE rats than in any other strain, and estradiol levels
were above detectable levels in F344 rats at this time. Serum
LH patterns were similar to those seen at the earlier time: HLZ
and F344 rats showed low levels, whereas SD and LE rats
showed significantly higher levels of LH. These differences in
hormone levels in control animals are a function not only of
strain but also of the time of euthanasia, suggesting another
caution for the design of toxicological studies that incorporate
the measurement of ovarian or pituitary hormones. It is possible that the differences in control hormone levels across strains
may play a role in the differential sensitivity of the various
strains to the effects of chemicals. For example, LE rats have
much higher levels of estradiol than other strains at 2100 h. A
connection may exist between this and the apparent resistance
of LE rats to effects of ATR on implantation.
Although previous work has suggested that ATR can suppress prl levels under specific conditions unrelated to pregnancy (Cooper et al., 2000; Simpkins et al., 1998), it is possible that the chemical may not be as effective in altering prl
when administered during early pregnancy. The only strain in
which ATR produced significant preimplantation loss was
F344, and in those animals no significant effect on progester-
142
CUMMINGS, RHODES, AND COOPER
FIG. 5. Effect of atrazine on serum luteinizing hormone. Diurnal dosing ⫽ 1400 h. Nocturnal dosing ⫽ 0200 h. HLZ, Holtzman; SD, Sprague Dawley; LE,
Long Evans; F344, Fischer 344 rats. Data are expressed as means ⫾ SE. Data marked with an asterisk (*) are statistically different from vehicle-treated controls
within each strain and dosing interval with a cutoff of p ⬍ 0.05.
one levels was observed. On the other hand, it is possible that
ATR indeed reduces prl during early pregnancy, but that the
minimal amount of prl necessary to sustain CL function and
implantation is so low that no outward adverse effect was
observed in conjunction with the hormonal effect.
In summary, exposure of rats to ATR during early pregnancy produced no adverse effects in LE or SD rats. HLZ and
F344 rats appear most sensitive to the effects of ATR on early
pregnancy. Our hypothesis that ATR should produce a preimplantation loss that might be based on the chemical’s effect on
prl has been shown to be true only for F344 rats. Postimplantation effects, likely to be mediated by LH, were produced by
ATR only in HLZ rats. We conclude that the effects of ATR on
early pregnancy may be selective with respect to strain and are
significant only in the sensitive strains. Further work to delineate effects of ATR on prl in susceptible strains during early
pregnancy is necessary.
REFERENCES
Barrow, D. L., and Tindall, G. T. (1983). Pituitary adenomas: An update on
their management with an emphasis on the role of bromocryptine. J. Clin.
Neuroophthalmol. 3, 229 –237.
Cooper, R. L., Parrish, M. B., McElroy, W. K., Rehnberg, G. L., Hein, J. F.,
Goldman, J. M., Stoker, T. E., and Tyrey, L. (1995). Effect of atrazine on the
hormonal control of the ovary. The Toxicologist 15, 294.
Cooper, R. L., Stoker, T. E., Goldman, J. M., Parrish, M. B., and Tyrey, L.
(1996). Effect of atrazine on ovarian function in the rat. Reprod. Toxicol. 10,
257–264.
Cooper, R. L., Stoker, T. E., Tyrey, L., Goldman, J. M., and McElroy, W. K.
(2000). Atrazine disrupts the hypothalamic control of pituitary-ovarian
function. Toxicol. Sci. 53, 297–307.
Cummings, A. M., Perreault, S. D., and Harris, S. T. (1991). Use of bromoergocryptine in the validation of protocols for the assessment of mechanisms
of early pregnancy loss in the rat. Fundam. Appl. Toxicol. 17, 563–574.
De Feo, V. J. (1967). Decidualization. In Cellular Biology of the Uterus (R. M.
Wynn, Ed.), pp. 191–290. Appleton-Century-Crofts, Meredith, NY.
ACKNOWLEDGMENTS
Greenwood, F. C., Hunter, W. M., and Glover, J. S. (1963). The preparation of
131
I-labeled human growth hormone of high specific activity. Biochem. J. 89,
114 –123.
The authors acknowledge the excellent technical assistance of Keith McElroy, Dorothy Guidici, and Joan Hedge for assistance with the hormone assays.
We also thank the National Hormone and Pituitary Agency for the gift of the
radioimmunoassay materials for LH.
Gressel, J., Ammon, H. U., Fogelfors, H., Kay, Q. O. N., and Kees, H. (1984).
Discovery and distribution of herbicide-resistant weeds outside of North
America. In Herbicide Resistance in Plants (H. M. LeBaron, J. Gessel,
Eds.), pp. 31– 46. John Wiley & Sons, New York.
ATRAZINE AND IMPLANTATION
Infurna, R., Levy, B., Meng, C., Yau, E., Traina, V., Rolofson, G., Stevens, J.,
and Barnett, J. (1988). Teratological evaluations of atrazine technical, a
triazine herbicide, in rats and rabbits. J. Toxicol. Environ. Health 24,
307–319.
Morishige, W. K., and Rothchild, I. (1974). Temporal aspects of the regulation
of corpus luteum function by luteinizing hormone, prolactin and placental
luteotrophin during the first half of pregnancy in the rat. Endocrinology 95,
260 –274.
Narotsky, M. G., Guidici, D. L., Best, D. S., Bielmeier, S. R., and Cooper,
R. L. (1999). Strain comparisons of susceptibility to pregnancy loss in rats.
Annual Meeting of the Endocrine Society, p. 492.
Peters, J. W., and Cook, R. M. (1973). Effects of atrazine on reproduction in
rats. Bull. Environ. Contam. Toxicol. 9, 301–304.
Psychoyos, A. (1973). Hormonal control of ovoimplantation. Vitamins and
Hormones 31, 201–256.
SAS Institute. (1985). SAS User’s Guide: Statistics, 5th ed. SAS Institute,
Cary, NC.
Simpkins, J. W., Eldridge, C., and Wetzel, L. T. (1998). Role of strain-specific
reproductive patterns in the appearance of mammary tumors in atrazinetreated rats. In Triazine Herbicides Risk Assessment (L. G. Ballantine, J. E.
McFarland, and D. S. Hackett, Eds.), pp. 399 – 413. American Chemical
Society, Washington, DC.
Sinclair, J. L., and Lee, T. R. (1992). Biodegradation of atrazine in subsurface
environments. Environmental Research Brief, EPA/600/S-92/001.
143
Smith, M. S., McLean, B. K., and Neill, J. D. (1976). Prolactin: the initial
luteotropic stimulus of pseudopregnancy in the rat. Endocrinology 98,
1370 –1377.
Stevens, J. T., and Sumner, D. D. (1991). Herbicides. In Handbook of Pesticide
Toxicology (W. J. Hayes and E. R. Laws, Eds.), Vol. 3, pp. 1317–1408.
Academic Press, NY.
Stevens, J. T., Breckenridge, C. B., Wetzel, L. T., Gillis, J. H., Luempert,
L. G., and Eldridge, J. C. (1994). Hypothesis for mammary tumorigenesis in
Sprague-Dawley rats exposed to certain triazine herbicides. J. Toxicol.
Environ. Health 43, 139 –153.
Terkel, J. (1988). Neuroendocrine processes in the establishment of pregnancy
and pseudopregnancy in rats. Psychoneuroendocrinology 13, 5–28.
Wetzel, L. T., Leumpert, L. G., Breckenridge, C. B., Tisdel, M. O., Stevens,
J. T., Thakur A. K., Extrom, P. J., and Eldridge, J. C. (1994). Chronic effects
of atrazine on estrus and mammary tumor formation in female Sprague
Dawley and Fischer 344 rats. J. Toxicol. Environ. Health 43, 169 –182.
Yochim, J. M., and De Feo, V. J. (1963). Hormonal control of the onset,
magnitude, and duration of uterine sensitivity in the rat by steroid hormones
of the ovary. Endocrinology 72, 317–326.
Zeleznik, A. J., and Fairchild-Benyo, D. (1994). Control of follicular development, corpus luteum function, and the recognition of pregnancy in higher
primates. In The Physiology of Reproduction, 2nd ed. (E. Knobil and J. D.
Neill, Eds.), pp. 751–782. Raven Press, Ltd., New York.