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*
Department of Anatomy and Developmental Biology and Congenital Anomaly Research Center, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan;
National Institute of Toxicology Research, Korea Food and Drug Administration, Seoul 122-704, Korea
3To whom correspondence should be addressed: Telephone, +81-75-753-4341; Fax, +81-75-751-7529; E-mail, kshiota{at}med.kyoto-u.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • folic acid • myo-inositol • hyperthermia • neural tube defects • pregnancy • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The two major types of NTD are anencephaly and spina
bifida, which are formed by the failure of closure of the anterior
(rostral) and posterior (caudal) neuropores, respectively, during the
fourth week of gestation. Some cases of human NTD are attributed to
pure genetic or environmental causes, but many cases of NTD are assumed
to be multifactorial in origin, i.e., due to an interaction of multiple
genes (polygenes) and environmental factors. This hypothesis is
supported by the fact that the frequency of anencephaly and spina
bifida varies with demographic factors such as the season and place of
birth, race, sex, social class, maternal age, and parity (Elwood and Elwood 1980
).
Recent clinical studies showed that a substantial proportion of NTD can
be prevented by folic acid (FA) supplementation prior to conception and
during the first months of pregnancy. In 1981, Smithells et al.
reported that women who had previously had a pregnancy affected by
spina bifida or anencephaly were given daily preparations of
multivitamins including FA and that they were seven times less likely
to have another affected pregnancy than were women who did not take the
vitamins. A randomized trial by the Medical Research Council of the UK
confirmed that the recurrence of NTD could be prevented by FA
supplementation (MRC Vitamin Study Research Group 1991
).
Studies that followed these trials showed that the first occurrence of
NTD is also reduced by periconceptional intake of FA, by approximately
60% (Czeizel and Dudas, 1992
, Werler et al. 1993
). In 1993, the U.S. Public Health Service issued the
recommendation that all women of child-bearing age who are capable
of becoming pregnant should consume 0.4 mg/day of FA (Centers for Disease Control 1993
).
The preventive effect of folate on NTD has been confirmed
experimentally (Zhao et al. 1996
). However, the animal
model of NTD they used was a special mutant that is deficient for the
Cart1 homeobox gene, and therefore it is not clear whether their data
can be extrapolated to NTD in general. Brief maternal hyperthermia (HT;
>2.5°C above normal) early in pregnancy was implicated in the
occurrence of NTD both in humans and laboratory animals, and the
pattern of malformation and the susceptibility to heat-induced
teratogenesis vary by animal species and strains (Edwards et al. 1995
). When pregnant mice were experimentally exposed to HT,
anterior NTD including exencephaly and anencephaly were induced
frequently (Shiota 1988
). Since the teratogenicity of HT
can be modified by other exogenous agents such as alcohol
(Shiota et al. 1988
), vitamin A (Ferm and Ferm 1979
), and lead (Edwards and Beatson 1984
), a
heat-induced NTD in rodents is thought to be a good experimental
model of multifactorial NTD. In the present study, we examined whether
FA supplementation can ameliorate the teratogenicity of HT in mice.
It has been postulated that about 30% of human NTD are
folate-resistant and cannot be prevented by FA supplementation.
Seller (1994)
and Greene and Copp (1997)
demonstrated that myo-inositol (MI), which plays a vital role in
the inositol/lipid cycle, reduces the incidence of NTD in curly tail
mice which are not prevented by FA or its metabolites. Therefore, the
effectiveness of MI supplementation to the HT-induced NTD was also
examined using our mouse model.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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FA, MI and other chemicals were purchased from Wako Pure Chemical Co. (Osaka, Japan) and were of the highest purity available.
Animals and diets.
ICR strain mice (SLC Japan, Hamamatsu, Japan) were maintained in a temperature- and humidity-controlled animal facility with a 12:12-h light/dark cycle. Animals were given free access to laboratory food (MFTM; Oriental Yeast Industry Co., Tokyo, Japan) and tap water. Of the diet 100 g included 0.14 mg of FA and 424 mg of inositol as components. Mature female mice usually eat 4–5 g of food daily, and their intake of FA per day was calculated to be 0.006–0.007 mg, which was considered to be low enough not to affect the study. The calculated daily inositol intake was 17–21 mg, and that of MI should have been less than the intake of inositol and was also considered to be low enough not to affect the study. Female mice (8–10 wk) were mated overnight. The females were checked for the presence of a vaginal plug on the next morning, and noon of the day on which a vaginal plug was found was considered gestational day (GD) 0.5. The females with vaginal plugs were divided into five groups of equal mean body weight. A total of 12 to 14 pregnant mice were assigned to each group.
Heat treatment.
When HT was induced on GD 8.5, anterior NTD were most frequently
induced, which was significantly higher than the corresponding figures
for the treatment on GD 7.5 and 9.5. At GD 8.5, the anterior neuropore
begins closing in the mouse embryo, which corresponds to d 22–24 after
fertilization in human gestation (O'Rahilly and Müller 1987
). Therefore, HT was induced in dams at GD 8.5, the most
susceptible stage to heat shock stress, by submerging the lower
two-thirds of the body in hot water as described previously
(Shiota 1988
). The water bath was equipped with a
thermoregulator (Taiyo Kagaku Kogyo, Tokyo) and a stirrer so that the
water temperature is maintained within a deviation of ±0.1°C from
the set temperature. HT lasted for 8.5 min at 43°C. Under the
condition of heat exposure which we used in the present study, the
incidence of dead and malformed fetuses increased significantly in a
dose-dependent manner, but beyond this condition the litters were
often dead or all the fetuses dead (Shiota 1988
).
Experimental design and teratological evaluations.
The experimental procedure is summarized in Figure 1.
To examine the effects of treatment with FA, each in a group of
randomly selected pregnant mice were given FA (3 mg/kg) dissolved in
physiological saline by gastric intubation at noon daily on GD 0.5
through GD 9.5. Another group of mice were similarly treated with FA
and then heated at GD 8.5 at 43°C for 8.5 min. For examining the
effects of treatment with MI, MI (500 mg/kg) was dissolved in
physiological saline, given once daily from GD 0.5 through GD 9.5 by
intraperitoneal injections and mice were heated at GD 8.5. The doses of
FA and MI were chosen according to the previous studies by Zhao et al. (1996)
and Greene and Copp (1997)
,
respectively. The vehicle control (VC) group for heat treatment was
given physiological saline during the same period (GD 0.5 through GD
9.5) and heated at GD 8.5. On GD 18.5, all the dams were killed by
cervical dislocation. The uteri were removed and numbers of
implantation sites, resorptions, and dead fetuses were recorded. Live
fetuses were sexed, weighed, and examined under a dissecting microscope
for external anomalies.
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Fetal data were analyzed by ANOVA with the Bonferroni post hoc test. Wilcoxon rank sum test was applied to compare the distributions of percentages between groups. The fetal data were analyzed on a litter basis to minimize the biases resulting from the litter effect. Statistical analysis was undertaken using a computer software program (StatView 4.01). The difference between groups was considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). When pregnant mice were heated at 43°C for 8.5 min at GD 8.5 of
gestation, 23.5% of the implanted conceptuses were resorbed,
indicating that they died early in the postimplantation period.
Treatment of pregnant females with FA (3 mg/kg) or MI (500 mg/kg) alone
exerted neither teratogenic nor embryolethal effects (Table 2
). None of their term fetuses were affected by NTD. Among the fetuses that
survived to term in the heat-treated group without FA or MI
pretreatment, 38.6% had cranial NTD (exencephaly and anencephaly).
When pregnant mice were pretreated with FA daily from GD 0.5 through GD
9.5 and heated at GD 8.5, the prevalence of NTD in the fetuses was
26.6%, which was significantly lower than the corresponding figure in
the VC + HT group (P < 0.05). Since morphological
abnormalities often cause embryonic death, the combined rates of
malformed fetuses and resorptions were compared between the groups. The
rate was 53.0% (88:166) for the VC + HT group and 40.9% (65:159)
for the FA + HT group (P < 0.05). The percentage
of dams with affected fetuses did not differ between the groups. When
pregnant mice were pretreated with MI and then heated, the prevalence
rate of NTD was 31.2%, which was not significantly different from the
corresponding value for the VC + HT group.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Holmes et al. 1976
).
In previous studies, FA antagonists, including methotrexate,
aminopterin, pyrimethamine and trimethoprim, have been associated with
various birth defects both in humans and experimental animals. These
drugs inhibit the enzyme dihydrofolate reductase. Aminopterin produces
a malformation syndrome that includes cranial dysplasia and other
craniofacial anomalies (Shaw and Steinbach 1968
,
Thiersch 1952
). Clinical doses of methotrexate early in
pregnancy have been implicated in some developmental anomalies and
spontaneous abortion (Milunsky et al. 1968
).
Experimentally, large doses of aminopterin and methotrexate produce
various malformations in laboratory animals, often involving the
central nervous system (Baranov 1966
, Skalko and Gold 1974
). Thus, it is reasonable to assume that inhibition of
folate metabolism can result in developmental abnormalities and that
folate supplementation during organogenesis may prevent embryos from
these birth defects. Recently, Pugarelli et al. (1999)
suggested that the efficacy of dietary FA supplementation in reducing
the incidence of NTD in human populations is related to methionine
metabolism. Folate deficiency decreases the embryo's supply of
methionine, and the regeneration of methionine from homocysteine,
following methyl group donation by S-adenosyl methionine, is
a folate-dependent reaction. Thus, the metabolic pathways of folate
and methionine are intimately interconnected, and methionine may well
be involved in the prevention of NTD by FA.
It has been suggested that human embryos need a maternal intake of
400–800 µg of folate to develop normally (Cziezel and Dudas 1992
, MRC Vitamin Study Research Group 1991
).
According to Hall (1997)
, our modern diet provides on
average, probably half of the FA + folate that we need to function
optimally. Clinical evidence has accumulated that folate
supplementation before conception and during early pregnancy can
prevent the recurrence as well as the first occurrence of NTD. However,
the mechanism by which folate prevents NTD is not well understood. One
of the reasons for this is the absence of good animal models.
Zhao et al. (1996)
prevented NTD in Cart1 mutant mice by
treating the dams with large doses of FA. However, the animals they
used were mutant with the Cart1 homeobox gene and therefore their
results may not be extrapolated to NTD in general, especially to those
of multifactorial origin. In addition, the pups in the
folate-supplemented group had anomalies other than NTD and usually
died in the perinatal period. Our present results provide evidence that
FA supplementation during early pregnancy can protect embryos against
NTD caused by exogenous factors or that are multifactorial in origin.
Under our experimental conditions, the prevalence of NTD in embryos
heated in utero was reduced to about two-thirds in the
folate-supplemented group. Since the percentage of females with
malformed fetuses did not differ between groups, FA treatment
apparently decreased NTD embryos uniformly in each litter. It is not
clear how FA can prevent heat-induced NTD in mice and this is now
under investigation in our laboratory. Seller (1994)
speculated that NTD could be produced by factors that result in
decreased cell proliferation. We found that a brief maternal HT can
cause a transient arrest of cell proliferation in neurulating mouse
embryos (Li and Shiota, unpublished). FA may possibly
ameliorate mitotic arrest of neural precursor cells in heated embryos
and reduce the risk of NTD.
Recently, Greene and Copp (1997)
reported that
supplementation of MI can prevent folate-resistant NTD. They
observed a major reduction in NTD in the offspring of curly tail mice
when pregnant dams were treated with MI during the critical period of
neural tube closure. The authors suggested that MI prevents the
occurrence of NTD by stimulating protein kinase C activity which, in
turn, enhances retinoic acid receptor ß expression, thereby
normalizing neurulation in the curly tail embryo. In our mouse model of
heat-induced NTD, the preventive effect of MI was not evident. It
may be worth investigating whether MI can prevent many of the
folate-resistant NTD or if its effect is limited to such NTD as
those seen in curly tail mutants.
Many interacting genes and environmental factors culminate in normal neural tube closure, but their roles in neural tube closure in human embryos are largely unknown. In the future, factors other than FA and MI may be identified that protect embryos against NTD. Further studies are required to elucidate the mechanisms of NTD prevention by vitamins and to determine the lowest doses that effectively prevent NTD in human embryos. At least for the time being, better nutritional intake during early pregnancy is recommended to decrease the birth of malformed children.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 The costs of publication of this article were defrayed in part by the payment of page charges. This article must
therefore be hereby marked "advertisement" in accordance with 18
USC section 1734 solely to indicate this fact.
4 Abbreviations used: FA, folic acid; GD, gestational day; HT, hyperthermia; MI, myo-inositol; NTD, neutral tube defects; VC, vehicle control.
Manuscript received June 3, 1999. Initial review completed June 25, 1999. Revision accepted August 5, 1999.
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REFERENCES |
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1. Baranov V. S. The specificity of the teratogenic effect of aminopterin as compared with other teratogenic agents. Bull. Exp. Biol. 1966;1:77-82(Russian)
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6. Elwood J. M., Elwood J. H. Epidemiology of Anencephalus and Spina Bifida. Oxford University Press, Oxford 1980;
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9. Hall J. G. Give the embryo a chance (News and Views). Nature Med 1997;3:24-25[Medline]
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) through GD 9.5 (
). HT was
induced at GD 8.5 (•). The dams were killed and fetuses observed at
GD 18.5 (
). Noon of the day the plug was found = GD 0.5.