Download Effects of Acetaminophen on Preimplantation Embryo Glutathione

56, 150 –155 (2000)
Copyright © 2000 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Effects of Acetaminophen on Preimplantation Embryo Glutathione
Concentration and Development in Vivo and in Vitro
Delia N. Laub, Nawal O. Elmagbari, Nura M. Elmagbari, Melissa A. Hausburg, and Catherine S. Gardiner 1
Department of Biology, University of Northern Colorado, Greeley, Colorado 80639
Received December 28, 1999; accepted February 25, 2000
This study investigated the effects of high doses of acetaminophen (APAP) on preimplantation embryos. Previous studies indicate that cleavage-stage embryos cannot synthesize reduced glutathione (GSH) de novo and may be sensitive to GSH-depleting
toxicants. Alternatively, there may be maternal mechanisms that
protect the embryos from the adverse effects of these toxicants. To
address these possibilities, we cultured two-cell stage embryos in 0,
375, 750, or 1500 ␮M APAP and evaluated GSH concentration
and development. APAP depressed embryo development to the
morula and blastocyst stages in vitro, but a decrease in embryo
GSH concentration was not detected. Furthermore, administration of 800 or 1430 mg/kg APAP to female mice 12 h prior to
embryo collection on day 2 of gestation, or administration of 800
mg APAP/kg/day from day – 8 to day 1 or day 3 of gestation, did
not significantly affect ovary or embryo GSH concentration or
embryo development. Liver GSH, however, was significantly decreased. Moreover, no adverse effects on embryo development to
term were observed after treatment of female mice with 1430 mg
APAP/kg/day from day – 8 to day 3 of gestation. In summary, in
vitro embryos were adversely affected, in terms of development, by
APAP. In vivo, large doses of APAP depleted liver GSH but did
not affect development of preimplantation embryos. In conclusion,
preimplantation embryos appear to be protected from GSH-depleting toxicants such as APAP in vivo.
Key Words: preimplantation; glutathione; acetaminophen; embryo.
Acetaminophen (APAP) is commonly used for its analgesic
and antipyretic properties and is very safe under normal conditions (Sipes and Gandolfi, 1991). In the liver, glucuronidation and sulfation metabolize APAP, with only a small amount
entering the P-450 oxygenase system. The P-450 system converts APAP into a highly reactive intermediate, N-acetylbenzoquinone imine (NAPQI), which can conjugate with reduced glutathione (GSH). A high dose of APAP will saturate
the glucuronidation and sulfation pathways and increase the
amount of APAP metabolized by the P-450 oxygenase system.
Consequently, NAPQI levels increase, leading to depletion of
hepatic stores of GSH. Eventually, NAPQI will bind with
1
To whom correspondence should be addressed. Fax: (970) 351-2335.
Email: [email protected].
cellular macromolecules, causing tissue necrosis and death.
Mitchell et al. (1973) administered one dose of 375 mg/kg
APAP ip to mice and found liver necrosis in 45% of the
animals examined. Furthermore, it has been demonstrated that
hepatoxicity increases when female mice are ovariectomized
(Raheja et al., 1983). Finally, the LD50 of APAP in mice has
been measured as 4.11% APAP or 5877 mg APAP/kg/day for
a 14-day exposure to APAP mixed with mouse chow (Reel et
al., 1992).
Earlier studies have shown that APAP can cause adverse
effects on embryos or fetuses at high doses. Mice exposed to
doses up to 1430 mg APAP/kg/day in their food, using a
continuous breeding protocol, exhibited a decrease in the number of litters per pair, post-natal weight gain, and a decrease in
birth weight of F2 pups (Reel et al., 1992). Stark et al. (1989a)
found open neural tubes in day 9 rat embryos cultured for 24 h
with 0.50 mM APAP, while Weeks et al. (1990) found open
neural tubes as well as incomplete body curvature in rat embryos cultured with 300 –750 ␮M APAP from days 9.5 to 11.5
of gestation. Furthermore, APAP overdoses in pregnant
women have led to birth defects and fetal death (Haibach et al.,
1984). However, the sensitivity of the preimplantation embryo
to APAP, both in vitro and in vivo, has not been previously
examined.
In the reduced form, the endogenous tripeptide, glutathione
(GSH, ␥-glutamycysteinylglycine), serves to protect cells from
oxidative stress, aids in metabolism of toxicants, and functions
as a transport form of cysteine (Reed, 1990). GSH also has an
important functional role in reproduction and early development. During normal fertilization, high levels of GSH must be
present in an oocyte for sperm nuclear decondensation to occur
(Perreault et al., 1988). When mice are treated with a GSH
synthesis inhibitor, L-buthionine S,R-sulfoximine, oocytes
showed a decrease in GSH content from 1.8 pmol to 0.20 pmol
GSH per ovum (Calvin et al., 1986). In addition, depletion of
GSH in mouse oocytes can adversely affect sperm nuclear
decondensation and microtubule function, which consequently
impairs embryonic development (Zuelke et al., 1997). During
normal embryo development, GSH concentration decreases
90% during the period from the unfertilized oocyte to the
blastocyst stage (Gardiner and Reed, 1994). In addition, em-
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EFFECTS OF ACETAMINOPHEN ON PREIMPLANTATION EMBRYOS
bryos cannot synthesize GSH de novo until the blastocyst
stage, which may leave them particularly susceptible to the
effects of toxicants (Gardiner and Reed, 1995). Recently, GSH
was discovered in the reproductive tract fluid of mice and was
found to improve development of preimplantation embryos
after chemically induced depletion of GSH (Gardiner et al.,
1998).
Because early embryos cannot synthesize GSH (Gardiner
and Reed, 1995), we hypothesize that they may be very sensitive to the effects of glutathione-depleting toxicants such as
APAP. An alternative hypothesis is that there are maternal
mechanisms that function to decrease exposure of embryos to
toxicants and to protect the embryos during preimplantation
development. We conducted 4 experiments to begin to address
both of these hypotheses. In the first experiment, we investigated the direct effect of APAP supplementation in culture
medium on preimplantation embryos. We also examined the
effects of one dose of APAP on the embryos, ovaries, and liver
of pregnant mice allowed to recover for 12 h in vivo. Next, we
determined the effects of daily APAP exposure in vivo
throughout preimplantation development on liver, ovary, and
embryo GSH, as well as embryo development at day 3 of
gestation. Finally, we investigated the effect of APAP on fetal
development to term when the dam was given APAP daily
from 8 days prior to ovulation until day 3 of gestation.
MATERIALS AND METHODS
Materials. Chemicals necessary for our experiments were purchased from
Sigma (St. Louis, MO) and mice were bred from NSA mice from Harlan
(Indianapolis, IN).
Treatment of mice with acetaminophen. The acetaminophen was suspended in a 0.5% tragacanth solution and given to female pubertal mice
intragastrically at 800 or 1430 mg/kg of body weight. The control group
received only the 0.5% tragacanth solution.
Embryo collection and evaluation. Female pubertal NSA mice were synchronized and superovulated by intraperitoneal injection with 10 IU equine
chorionic gonadotropin and 44 – 48 h later with 5 IU of human chorionic
gonadotropin. Females were bred with proven breeder males and were checked
the next day for a copulation plug (designated as day 0 of gestation). Females
were euthanized on day 1 (d1), day 2 (d2), or day 3 (d3), and 2-cell, morula,
or blastocyst stage embryos were collected by flushing M16 medium through
the oviducts and/or uteri (Hogan et al., 1994). Embryos were then washed by
sequential passage though drops of M16 medium with 4 mg/ml bovine serum
albumin (BSA). Embryos cultured in vitro were placed into 10 ␮l drops (10
embryos per drop) of M16 medium with 4 mg/ml BSA and covered with
mineral oil. Embryos were cultured in a humidified atmosphere of 5% CO 2 in
air at 37°C. Daily microscopic evaluation of embryos consisted of noting
fertilization, classifying embryos into their respective developmental stages,
and identifying degenerate embryos. Degenerate embryos were not used for
GSH analysis.
Liver and ovary collection and evaluation. Portions of the liver and both
ovaries were dissected from euthanized female mice and weighed. The overall
morphology of the tissues was assessed, and the samples were prepared for
quantification of GSH.
Fetus collection and evaluation. Female pubertal mice were bred with
proven breeder males on day –1. On d17 of gestation, dams were euthanized
and fetuses were removed via cesarean section. Maternal and fetal evaluation
151
FIG. 1. Embryo development in vitro when cultured with APAP from the
2-cell stage (d1). Developmental stages are indicated as follows: deg (degenerate), 4 – 8 (4-cell to 8-cell), M (compacted morula), B (blastocyst), XB
(expanded blastocyst), IH (initiating hatching blastocyst), and H (hatched
blasto cyst). Within a developmental stage, columns not accompanied by the
same letter (a,b,c,d) are significantly different (p ⬍ 0.05). Values are means ⫾
standard error.
consisted of recording the number of resorptions in the uterus, weighing the
intact uterus with the fetuses, weighing the pregnant dam, examining each
fetus for gross malformations, weighing individual fetuses, and noting the
number of pups per litter.
Detection of GSH and GSSG by HPLC. Pools of 20 –30 embryos, liver
samples, and ovary samples were homogenized in perchloric acid-diethylenetriaminepentaacetic acid solution and prepared for GSH and GSSG detection
via high performance liquid chromatography (Martin and White, 1991). Each
sample was fluorescently labeled with dansyl chloride and subsequently analyzed on an amino propyl silica column using a methanol-sodium acetate
gradient system (Reed et al., 1980). The detection limit for this method is 1
pmol of GSH.
Effects of APAP in culture medium on embryo development and GSH
concentration in vitro. Embryos were collected on d1, washed, and placed
into culture drops at the 2-cell stage with 0, 375, 750, or 1500 ␮M APAP in
M16 with 4 mg/ml BSA. Embryos were derivatized on d2 (at the 4-cell to
compacted morula stage) for GSH quantification, and development was assessed on d2, d3, d4, and d5.
Effects of one dose of APAP on GSH concentration and embryo development in vivo. Mice found to have copulation plugs on d0 were treated on d2
with 0, 800, or 1430 mg APAP/kg. Mice were euthanized, and samples were
collected 12 h after dosing.
Effects of APAP treatment for 10 or 12 days on GSH concentration and
embryo development in vivo. Female mice were treated with 0 or 800 mg/kg
APAP daily from 8 days prior to ovulation until the morning of d1 or d3 of
gestation. Embryos and other tissues were collected on the afternoon of d1 or
d3.
Effects of preimplantation exposure with APAP on development to term.
Female mice were treated with 0 or 1430 mg/kg APAP daily starting 8 days
prior to ovulation and continuing until 3 days after copulation plugs were
detected. Fetuses were removed by cesarean section on d17 of gestation.
Statistical analysis. Morphological development was analyzed using
Kruskal-Wallis analysis of variance. Analysis of variance and least-significantdifference procedures were used to determine differences in GSH and GSSG
content of embryos, liver, and ovaries. A t-test was used to analyze the number
of fetuses per dam, number of resorptions per dam, individual fetal weight,
total fetal weight, uterine weight, liver weight, and weight of dam on d17.
Chi-square analysis was used to analyze the number of mice with copulation
plugs and the number of deceased mice. Statistica software was used for
statistical analysis. All experiments were repeated at least twice with a minimum of 3 replications for each treatment each time.
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LAUB ET AL.
FIG. 2. Embryo GSH after treatment in vitro with APAP for 24 h.
Embryos were at the 4-cell to compacted morula stage. No significant differences were detected. Values are means ⫾ standard error.
RESULTS
Effects of APAP in culture medium on embryo development
and GSH concentration in vitro. Embryos collected on d1 of
gestation (2-cell stage) and cultured with varied concentrations
of APAP did not develop to later stages as well as embryos
cultured in M16 with BSA alone (p ⬍ 0.05, Fig. 1). However,
GSH concentration on day 2 (at the 4-cell to compacted morula
stage) did not decrease to correspond with the lack of development in the groups of embryos treated with APAP (Fig. 2).
Effects of one dose of APAP on GSH concentration and
embryo development in vivo. Administration of one dose of
APAP intragastrically to pregnant female mice on d2 of gestation significantly decreased liver GSH concentrations (p ⬍
0.05, Fig. 3). Ovary and embryo GSH concentrations as well as
embryo development (Fig. 4) did not significantly differ due to
treatment with APAP.
Effects of APAP treatment for 10 or 12 days on GSH
concentration and embryo development in vivo. Figures
5 and 6 demonstrate that liver GSH concentration significantly
FIG. 3. GSH concentrations 12 h after treatment with APAP on d2 of
gestation. Within a tissue type, columns not accompanied by the same letter
(a,b) are significantly different (p ⬍ 0.05). Values are means ⫾ standard error.
FIG. 4. Embryo development in vivo 12 h after treatment with APAP on
d2 of gestation. No significant differences were detected. Values are means ⫾
standard error.
decreased due to treatment with APAP (p ⬍ 0.05); however,
ovary and embryo GSH concentrations as well as embryo
development (Figs. 7 and 8) were not affected.
Effects of preimplantation exposure with APAP on development to term. To assess how exposure during preimplantation
development affects fetal development to term, we exposed
female mice to APAP from 8 days prior to ovulation until day
3 of pregnancy and allowed embryos to develop until d17 in
vivo. Upon evaluation of the females on d17 of gestation,
maternal body weight did not differ significantly, nor did liver
or uterine weight (Table1). However, significantly fewer female mice were found with copulation plugs each day of
breeding in the 1430 mg/kg APAP group (p ⬍ 0.05; Control
18 ⫾ 4% plugged each day, APAP 4 ⫾ 2%). While no mice
FIG. 5. GSH concentration after daily APAP treatment from 8 days prior
to ovulation until the first day of gestation. Within a tissue type, columns not
accompanied by the same letter (a,b) are significantly different (p ⬍ 0.05).
Values are means ⫾ standard error.
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EFFECTS OF ACETAMINOPHEN ON PREIMPLANTATION EMBRYOS
FIG. 6. GSH concentration after daily APAP treatment from 8 days prior
to ovulation until d3 of gestation. Within a tissue type, columns not accompanied by the same letter (a,b) are significantly different (p ⬍ 0.05). Values are
means ⫾ standard error.
died in the control group, 9 mice treated with APAP died
during the course of the study. No gross malformations were
found, but individual fetuses from females treated with APAP
weighed significantly more than fetuses from the control dams.
DISCUSSION
Previous studies have shown that embryos do not possess the
capacity to synthesize glutathione de novo until the blastocyst
stage of development (Gardiner and Reed, 1995). Furthermore,
preimplantation embryos exhibit a 90% decrease in glutathione
concentration from the unfertilized oocyte to the blastocyst
stage (Gardiner and Reed, 1994). This may leave preimplantation embryos susceptible to the effects of toxicants such as
APAP that are normally detoxified by GSH. Alternatively,
FIG. 8. Embryo development after treatment with APAP from 8 days
prior to ovulation until day 3 of gestation. No significant differences were
detected. Values are means ⫾ standard error.
there may be maternal mechanisms present to protect the
embryos during this sensitive period.
Our first experiment demonstrated that development to the
morula and blastocyst stages was depressed in embryos cultured in 375, 750, and 1500 ␮M APAP. Previous studies have
demonstrated that APAP inhibits Na ⫹/K ⫹-ATPase (Corcoran
et al., 1987), an enzyme important in blastocoel formation
(Gardiner and Menino, 1993). This may partially explain our
observed decrease in embryo development to later stages.
Moreover, our results are consistent with studies of the effects
of APAP on postimplantation development of rat embryos. Rat
embryos cultured in APAP (0.50 mM) on d9, d10, or d11 of
gestation exhibit an increased incidence of neural tube defects
(Stark et al., 1989b) and decreases in embryonic length and
yolk sac protein (Stark et al., 1989a). Furthermore, previous
TABLE 1
Effects of Daily Dosing with APAP from Day ⴚ8 to Day 3 of
Gestation on Development to Term
Number of females
Weight of dam on d17 (g)
Number of mice with copulation plugs
Weight of liver (g)
Uterine weight (g)
Number of fetuses/dam
Number of resorptions/dam
Individual fetal weight (g)
Total fetal weight (g)
Number of malformations
FIG. 7. Embryo development after treatment with APAP from 8 days
prior to ovulation until the first day of gestation. No significant differences
were detected. Values are means ⫾ standard error.
0 mg/kg/day
1430 mg/kg/day
36
50.1 ⫾ 1.2
25*
3.0 ⫾ 0.1
15.6 ⫾ 1.1
11.7 ⫾ 0.9
1.5 ⫾ 0.6
0.90* ⫾ .01
10.5 ⫾ 0.9
0
36
49.7 ⫾ 2.6
8*
3.2 ⫾ 0.1
13.8 ⫾ 2.2
9.7 ⫾ 1.9
2.8 ⫾ 1.0
1.01* ⫾ 0.02
9.7 ⫾ 1.6
0
Note. Values are means ⫾ standard error unless otherwise indicated.
* Values significantly different from each other ( p ⬍ 0.05).
154
LAUB ET AL.
studies indicate that depletion of embryonic GSH concentration prior to APAP treatment causes an increase in the incidence of embryonic defects (Stark et al., 1989b), while N-acetylcysteine protects cultured d9 rat embryos from the
embryotoxic effects of 0.30 mM APAP in vitro (Weeks et al.,
1990).
At high doses, cytochrome P-450 monooxygenases convert
APAP to its reactive metabolite, NAPQI, which subsequently
depletes GSH, leading to toxicity. Previous studies have detected cytochrome P-450 activity in preimplantation embryos
at the blastocyst stage (Pedersen et al., 1985). Cytochrome
P4501A1 gene expression has also been detected as early as the
oocyte stage (Dey and Nebert, 1998). If the monooxygenase
system is functioning during preimplantation development,
APAP could be metabolized to NAPQI, causing a decrease in
GSH concentration. Our data did not show a significant decrease in embryo GSH at d2 of gestation after 24 h of culture
in APAP when embryos were at the morula stage. This may be
evidence of APAP toxicity without GSH depletion. It is also
possible that GSH levels had already recovered or GSH was
not yet depleted at the time of our measurement. Alternatively,
differences in GSH concentration due to APAP may have been
present but were not detectable due to variation in samples.
Previous studies show that toxicants can induce GSH synthesis
by increasing transcription of the catalytic subunit of the ratelimiting enzyme in GSH synthesis (Shi et al., 1994). APAP
could have induced early GSH synthesis, which would have
allowed the embryos to recover their intracellular GSH. Alternatively, embryo development could have been adversely affected by means other than depletion of GSH pools.
After the direct effects of APAP were assessed, we examined the effects of APAP in vivo. APAP was administered to
female mice, and effects were examined 12 h after one dose or
after 10 to 12 days of daily dosing. No significant differences
in ovary GSH concentration, embryo GSH concentration, or
embryo development were observed. Similar to previous studies (Mitchell et al., 1973), however, liver GSH concentration
decreased significantly following one or several doses of
APAP. Earlier studies indicate that hepatoxicity increases in
pregnant mice (Larrey et al., 1986). These data confirmed that
the doses of APAP administered were high enough to deplete
GSH. These results suggest that the preimplantation embryo is
at least partially protected from APAP in vivo and that the
reproductive tract does not experience significant GSH depletion due to the doses examined. One likely mechanism of
protection is the hepatic metabolism of a significant amount of
the APAP, which resulted in decreased exposure of the preimplantation embryo to the APAP. If the concentration of APAP
in the reproductive tract fluid were the same as in the in vitro
studies, the embryos may have exhibited similar adverse effects in vivo as were seen in vitro.
To determine whether exposure of embryos to APAP in vivo
during the preimplantation period leads to toxic effects on
development to term, female mice were treated with 1430
mg/kg/day APAP 8 days prior to ovulation and throughout
development to the blastocyst stage. Subsequently, effects on
fetuses at term were examined. Table 1 shows that weight of
dams on d17, liver weight, uterine weight, number of fetuses
per dam, number of resorptions per dam, and total fetal weight
were not significantly different. However, individual fetal
weights were higher in dams treated with APAP. This effect
may be due to the presence of a smaller number of fetuses per
dam and a higher number of resorptions in the uterus. Although
there was a trend for an increased number of resorptions and a
decreased number of fetuses in APAP-treated dams, the effect
was not significant. Furthermore, we did not detect any gross
malformations of fetuses in either group. Similarly, Lum and
Wells (1986) found that administration of one ip dose of APAP
does not cause cleft palate in mice. However, 9 out of 36 mice
in our APAP group died during the course of treatment, indicating that a toxic dose of APAP was administered. We found
significantly fewer female mice with copulation plugs in the
APAP group. It is not clear whether this was a physiological
effect causing a delay or absence of estrus and ovulation or an
effect on copulation behavior. Reel et al. (1992) also treated
mice with doses of APAP up to 1430 mg/kg administered in
mouse chow, using a continuous breeding protocol, and found
a decrease in the number of litters per pair, an increase in the
presence of abnormal sperm, and a decrease in post-natal
weight of the pups. Our results would suggest that these effects
were not due to toxicity at the preimplantation stages of development.
We conclude that preimplantation embryos are very sensitive to APAP toxicity in vitro. In vivo, however, there appear
to be maternal mechanisms protecting the embryos or decreasing the exposure of the embryos to APAP. The liver may
metabolize and conjugate enough APAP such that only very
low levels reach the reproductive tract. In support of this
hypothesis, our study did not detect a significant decrease in
ovarian GSH concentration. Furthermore, toxic concentrations
of APAP may not enter the reproductive tract fluid in the lumen
of the uterus where the preimplantation embryos are developing. Another possibility is that uterine fluid may harbor substances that protect the preimplantation embryo, as demonstrated by a previous study indicating that GSH is present in
uterine and oviductal fluid and has beneficial effects on preimplantation development (Gardiner et al., 1998). Further studies are required to elucidate the maternal mechanisms that are
protecting the embryos from the adverse effects of high doses
of APAP.
ACKNOWLEDGMENTS
The authors would like to thank James Salmen, Gene Gushansky, Shawn
Stover, Analouise Matt, and Leslie Calaustro for their assistance. This work
was supported by the National Institute of Environmental Health Sciences,
NIH grant ES08818.
EFFECTS OF ACETAMINOPHEN ON PREIMPLANTATION EMBRYOS
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