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Vol. 297, Issue 2, 774-779, May 2001
Section on Developmental and Molecular Pharmacology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (C.Y.S., D.T.A., D.E.B., J.M.H.); and Department of Clinical Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel (I.G.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Two peptides [NAPVSIPQ (NAP) and SALLRSIPA (ADNF-9)], that are associated with novel glial proteins regulated by vasoactive intestinal peptide, are shown now to provide protective intervention in a model of fetal alcohol syndrome. Fetal demise and growth restrictions were produced after intraperitoneal injection of ethanol to pregnant mice during midgestation (E8). Death and growth abnormalities elicited by alcohol treatment during development are believed to be associated, in part, with severe oxidative damage. NAP and ADNF-9 have been shown to exhibit antioxidative and antiapoptotic actions in vitro. Pretreatment with an equimolar combination of the peptides prevented the alcohol-induced fetal death and growth abnormalities. Pretreatment with NAP alone resulted in a significant decrease in alcohol-associated fetal death; whereas ADNF-9 alone had no detectable effect on fetal survival after alcohol exposure, indicating a pharmacological distinction between the peptides. Biochemical assessment of the fetuses indicated that the combination peptide treatment prevented the alcohol-induced decreases in reduced glutathione. Peptide efficacy was evident with either 30-min pretreatment or with 1-h post-alcohol administration. Bioavailability studies with [3H]NAPVSIPQ indicated that 39% of the total radioactivity comigrated with intact peptide in the fetus 60 min after administration. These studies demonstrate that fetal death and growth restriction associated with prenatal alcohol exposure were prevented by combinatorial peptide treatment and suggest that this therapeutic strategy be explored in other models/diseases associated with oxidative stress.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In
the United States, fetal alcohol syndrome (FAS) occurs in 0.5 to 3 births per 1000 births each year (Stratton et al., 1996
). During the
1990s, the prevalence of alcohol consumption, including binge drinking,
has increased among pregnant women, whereas there has been little
change in nonpregnant women of child-bearing age (Ebrahim et al.,
1999). Maternal alcohol consumption is the most commonly identifiable
nongenetic cause of mental retardation (Windham et al., 1997). In
addition, consuming more than three drinks per week in the first
trimester has been shown to double the risk of miscarriage (Jimenez et
al., 1993). Children with FAS have a characteristic set of facial
features, as well as craniofacial dysmorphology, microcephaly, growth
restriction, and central nervous system impairment. FAS, however, does
not represent all children with prenatally derived alcohol-induced
fetal injury, because the absence of the facial features precludes
assigning the diagnosis. Children with fetal alcohol exposure without
FAS can have serious mental and learning impairments and by magnetic
resonance imaging have evidence of structural brain defects (Mattson et
al., 1996).
The purpose of the present study was to attempt an intervention against
alcohol-induced damage by treating pregnant mice with peptides that
have been shown to be neuroprotective in dissociated cerebral cortical
cell culture (Brenneman et al., 1998; Bassan et al., 1999) and in vivo
(Bassan et al., 1999; Gozes et al., 2000). In the adult, there are
documented interactions between alcohol and vasoactive intestinal
peptide (VIP) (Gressens et al., 1992; Madeira et al., 1997), a
neuropeptide that is a regulator of early postimplantation mouse
embryonic growth (Spong et al., 1999). In addition, VIP antagonist
treatment of pregnant mice results in some of the features of FAS,
including fetal growth restriction and microcephaly (Gressens et al.,
1994). The rationale for the use of these peptides resides in the
neuroprotective (Brenneman and Eiden, 1986; Brenneman et al., 1988) and
growth-promoting (Gressens et al., 1993) actions of VIP. These actions
of VIP are mediated indirectly through the release of glia-derived
substances (Brenneman et al., 1987), including activity-dependent
neurotrophic factor (ADNF), a protein that has growth-promoting
(Glazner et al., 1999b), antiapoptotic (Gozes et al., 1997), and
antioxidative properties (Brenneman and Gozes, 1996). Analysis of
digest peptides of purified ADNF revealed active peptide fragments
(Brenneman and Gozes, 1996). Structure-activity analysis of an active
peptide fragment demonstrated that a nine amino acid peptide
(SALLRSIPA) was the shortest sequence that retained the potent,
protective properties of ADNF (Brenneman et al., 1998). This peptide
was named ADNF-9 because it was comprised of nine amino acids and captured the survival-promoting activity of ADNF.
Antibodies against ADNF-9 were used to clone and identify a
functionally related protein: activity-dependent neuroprotective protein (ADNP) (Bassan et al., 1999; Zamostiano et al., 2001). An ADNF-9-like fragment of ADNP [NAPVSIPQ (NAP)] was discovered that
protected against oxidative stress (Offen et al., 2000; Steingart et
al., 2000). ADNF-9 and NAP exhibit remarkable potency and
neuroprotective activity in animal models related to neurodegeneration,
including apolipoprotein E-deficient mice (Bassan et al., 1999), rats
treated with a cholinotoxin (Gozes et al., 2000), and mice subjected to closed-head injury (Beni-Adani et al., 2001). Thus, ADNF-9 and NAP were chosen for evaluation in a FAS model.
The model for FAS employed in the present study is based on an acute
high exposure to alcohol that provided an outcome emphasizing the
severity of alcohol-induced growth restriction and fetal death (Webster
et al., 1980). Since treatment on gestational day 8 resulted in the
highest rate of fetal anomalies and demises (Webster et al., 1980), and
VIP's growth-regulating effects on the embryo are limited to the early
postimplantation period (Gressens et al., 1993; 1994), day 8 was chosen
as the optimal and most severe test for the protective activity of the
peptides. For accurate and reproducible administration of alcohol and
peptide, a model utilizing intraperitoneal injection was chosen
(Webster et al., 1980). The highest alcohol dose in the model (0.03 ml/kg) was selected to provide the most severe test of efficacy.
Previous studies indicated that the intraperitoneal model results in
higher blood alcohol concentrations than that obtained by an oral route (Webster et al., 1980), providing a stringent test to evaluate treatment efficacy.
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Materials and Methods |
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Introduction
Materials and Methods
Results
Discussion
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Animals. C57-Bl6J female mice (Harlan Sprague-Dawley, Inc., Indianapolis, IN) were kept under a 12-h light/dark regimen, with food and water available at all times. The mice received humane animal care in compliance with the Guideline for Care and Use of Experimental Animals. Six-week-old females (21-24 g) were mated with C57-Bl6J males for 4 h. The presence of a vaginal plug was considered day 0 of pregnancy.
Treatment Groups.
A well delineated model for FAS was used
(Webster et al., 1980). Animals were injected (intraperitoneal; i.p.)
on gestational day 8 with 25% ethyl alcohol in saline (v/v) or vehicle
alone at 0.030 ml/g of body weight at 9 AM. Pretreatment (i.p.) with VIP and ADNP/ADNF peptides (NAP, ADNF-9, NAP + ADNF-9) were given 30 min before alcohol. Post-treatment with NAP + ADNF-9 was given at 1 or
3 h after alcohol. Dosages of the peptides were NAP (20 and 40 µg), ADNF-9 (20 µg), NAP (20 µg) + ADNF-9 (20 µg), and VIP (1 µg). NAP + ADNF-9 without alcohol was also studied. NAP was diluted
in 50 µl of dimethyl sulfoxide and diluted in filtered Dulbecco's
phosphate-buffered saline (DPBS). ADNF-9 was dissolved and diluted in
filtered DPBS. VIP was dissolved in 20 µl of acetic acid (0.02%) and
diluted in water and DPBS. Since the animals receiving alcohol were
incapacitated for approximately 6 h following injection, food and
water were withheld from all groups for the initial 6 h to allow
accurate assessment of fetal weights.
Evaluation of Litter Size and Pup Weights. Fetal brain and body weights were obtained on pregnancy day 18. The number of pups and fetal deaths/resorption were counted. The number of live pups was calculated as a percentage of the total litter size. The litter mean was used as the N for comparisons of fetal survival, pup weight, and brain weight.
Bioavailability.
The bioavailability of NAP to embryos was
assessed by intraperitoneal administration of
[3H]NAP (3 µCi/20 µg of NAP/injection) to
pregnant mice at E8. Before labeling with tritium, NAPVSIPQ was
synthesized with one modification: replacing the proline in the third
position with a dihydroproline (SynPep Corp., Dublin, CA). The NAPVSIPQ
[propyl 3,3,4-3H] was custom-labeled by
American Radiolabeled Chemicals, Inc. (St. Louis, MO). The specific
activity of the [3H]NAP was 50 µCi/mmol, and the
labeled peptide was dissolved in ethanol and stored at 
80°C. The
purity of the labeled NAP was >99.5% as determined by
high-performance liquid chromatography with a Vydac C18 column
(4.6 × 250 mm). Tissues were collected 30 and 60 min after
injection into 300 µl of 0.1 N HCl. Samples were homogenized with
disposable pellet pestles and centrifuged at 10,000g (10 min
at 4°C). The supernatant was frozen on dry ice and stored at 80°C
until analyzed by size exclusion analysis (Superdex Peptide HR 10/30;
Amersham Pharmacia Biotech, Piscataway, NJ) on a fast performance
liquid chromatograph.
Reduced (GSH) and Oxidized (GSSG) Glutathione Levels.
GSH
and GSSG were simultaneously detected and measured with capillary
electrophoresis in embryo/decidua 8 h after treatment using a
previously described method (Muscari et al., 1998). NAP + ADNF-9 was
given 30 min before alcohol. Embryo/decidual samples were pooled in
groups of two; at least eight samples were run per treatment. Identity
of GSH and GSSG was made in relation to the migration of the
extraosmotic flow. Purified GSH and GSSG were added to similar
biological samples to confirm the chromatographic mobility (Sigma, St.
Louis, MO) and to verify the identity of the observed peaks.
Statistics.
The mean litter pup and fetal brain weight was
calculated, with the litter mean used for all statistical analysis.
Percent reabsorptions were calculated by dividing the number of
reabsorptions by the total number of fetuses (live + reabsorptions).
Statistical analysis included ANOVA for continuous variables with
Duncan's New Multiple Range test, Mann-Whitney U test for
nonparametric data, 
2 test for categorical
variables, or Fisher's Exact test where appropriate [Statview 4.5 (Abacus Concepts, Inc., CA)], with p < 0.05 considered significant.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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NAP + ADNF-9 Treatment Prevent Alcohol-Induced Fetal Death.
Prenatal administration with NAP + ADNF-9 to alcohol-treated pregnant
mice significantly increased the number of surviving fetuses in
comparison with those given alcohol alone (Fig.
1). As previously described (Webster et
al., 1980), alcohol treatment on gestational day 8 results in more than
one-third of the fetuses dying in utero (Fig.
2). Pretreatment (30 min before alcohol
administration) with NAP or the combination of NAP + ADNF-9 resulted in
a number of surviving fetuses that were not different than that of
control. Furthermore, these protective effects on fetal death were
observed even when the peptides were administered 1 h after
treatment with alcohol. NAP, alone or in combination with ADNF-9, was
required for the prevention of alcohol-induced fetal death. However, a 3-h post-treatment with the peptides did not result in a significant change in the number of surviving fetuses in comparison with the alcohol-treated group. In addition, treatment with NAP + ADNF-9 without
alcohol cotreatment resulted in an incidence of fetal death that was
not different from that of control (Fig. 2). Pretreatment with the
neurotrophic peptide VIP did not prevent the alcohol-induced fetal
death. The litter size (living + demises) was not different between the
groups, with an average litter size of eight fetuses. The number of
fetuses per treatment groups ranged between 140 and 400, and the litter
mean was used for statistical analysis.
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Combination of NAP + ADNF-9 Prevented Alcohol-Induced Fetal Growth
Abnormalities.
Both fetal brain and body weights were
significantly smaller in the litters from alcohol-treated mice,
compared with controls (Figs. 1 and 3). The effect of alcohol on body
size shown in Fig. 1 depicts the maximal effect observed in the study.
The summary of the effects on mean body weight for the various
treatment groups is shown in Fig. 3.
ANOVA analysis of these data indicated significant effects of alcohol
treatment and significant improvements with peptide administration.
With respect to changes in the mean body weight, pretreatment with NAP + ADNF-9 prevented the growth restrictions associated with alcohol.
Although neither peptide alone prevented alcohol-induced fetal growth
abnormalities, the combination of the two was effective. This effect
was not due to concentration, because pretreatment with a double dosage
of NAP (40 µg) did not prevent the alcohol-induced growth
restriction. This observation indicates that the combination of the two
peptides was necessary to prevent alcohol-induced growth restrictions.
Post-treatment with NAP + ADNF-9 (1 and 3 h) prevented the
microcephaly, but not the growth restriction. Pretreatment with VIP did
not prevent the alcohol-induced growth restrictions, and treatment with
NAP + ADNF-9 without alcohol resulted in fetal weights that were not different than that of control (Fig. 3, A and B). Treatment with the
peptides without alcohol resulted in body weights that were not
different from those of controls. Average litter size was 8 to 10 fetuses, thus there were approximately 110 to 330 fetuses in each
group.
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Bioavailability of NAP.
The biodistribution of
[3H]NAP was studied after intraperitoneal
injection of pregnant mice. These studies demonstrated that 68% of the
radioactivity recovered in the embryo comigrated with intact NAP 30 min
after administration (Fig. 4). Based on
amount of radioactivity and the specific activity of the peptide, the calculated average accumulation of labeled peptide was approximately 0.2 pmol of NAP in the embryo, with an estimated concentration of 10 nM, well within the therapeutic range of NAP as determined from in
vitro studies (Bassan et al., 1999). Analysis of the
[3H]NAP associated with the embryos 60 min after
injection indicated 39% comigrated with intact NAP.
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Prevention of Oxidative Stress.
Alcohol treatment resulted in
a significant decline of GSH/GSSG to 50% of control (p
<0.005). Pretreatment with NAP + ADNF-9 (20 µg each) prevented the
alcohol-induced decline in GSH/GSSG, with a ratio not different from
that of the control (Fig. 5)
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Systemic administration of two peptides, NAPVSIPQ and ADNF-9, has been shown to prevent alcohol-induced fetal demise and growth restrictions in a model of FAS. The results of this study clearly indicated that treatment with NAP alone was effective in preventing alcohol-induced fetal death, whereas ADNF-9 given alone was not protective. Cotreatment with both peptides was necessary for the prevention of growth restriction produced by prenatal alcohol treatment. Increasing the concentration with a double dosage of NAP did not prevent the alcohol-induced growth restriction; whereas adding ADNF-9 produced a significant effect on growth restriction. Since the combinatorial peptide treatment was initiated 10 days before evaluation, these studies indicate a prolonged period of protection that was elicited by peptides. However, a period of effective intervention was apparent from the studies. Prevention of alcohol-induced fetal death occurred when the peptides were administered 1 h after alcohol administration. Thus, intervention in the severe toxicity induced by alcohol was effective even after a substantial delay in the onset of peptide treatment. However, delaying the peptide treatment to 3 h after alcohol treatment resulted in no demonstrable protection. These studies suggest that a period of irreversible damage occurs with alcohol toxicity, but significant long-term protection can be obtained with early treatment with NAP/ADNF-9.
Although the mechanism(s) of action of these peptides are not fully
understood, the rationale for testing these drugs in the alcohol
paradigm was due to their demonstrated effects in preventing oxidative
damage and neuronal cell death in cell culture (Brenneman and Gozes,
1996; Bassan et al., 1999; Offen et al., 2000; Steingart et al., 2000).
Although toxicity associated with ethanol is complex, oxidative damage
due to the generation of free radicals and diminished reduced-glutathione appear to be important mechanistic components (Lieber, 1999; Mitchell et al., 1999). Peptide-mediated effects on
oxidative stress have been identified through the inhibition of
reactive oxygen species and mitochondrial peroxynitrite (Glazner et
al., 1999a). Therefore, the potential of these peptides in preventing
alcohol-induced damage appeared plausible. As an index of oxidative
stress (Devi et al., 1993), both reduced (GSH) and oxidized (GSSG)
glutathione were measured in gestations (embryo + decidua) 8 h
after treatment (Muscari et al., 1998). As previously shown (Devi et
al., 1993), alcohol treatment significantly decreased the ratio of
GSH/GSSG. Importantly, treatment with the peptides prevented the
alcohol-induced changes in reduced to oxidized glutathione levels in
the embryo/deciduas and resulted in a ratio not different from that of
control. The prevention of the alcohol-induced alteration of the
GSH/GSSG ratio supports the conclusion that alcohol treatment does
produce significant oxidative stress as assessed by this marker and
that the peptides act as oxidative protectants that are mediated in
part through glutathione. Other models support this conclusion: for
example, treatment with antioxidative agents, such as vitamin E or beta
-carotene, prevented alcohol-induced apoptotic cell death in cell
culture (Mitchell et al., 1999). In related studies of NAP and
ADNF-9-mediated protection from dopamine toxicity, a
glutathione-related mechanism appears to be involved (Offen et al.,
2000). Furthermore, toxicity associated with buthionine sulfoximine, a
selective inhibitor of glutathione synthesis, was significantly
attenuated with NAP treatment of neuroblastoma cells. Although the
mechanism(s) of alcohol-induced damage and death are incompletely
understood, it appears likely that a dysregulation of the oxidative
state occurs after alcohol administration and that NAP/ADNF-9 treatment
has an effect on an important regulator of oxidative homeostasis: glutathione.
Although the antioxidative properties of the peptides may play a major
role in their mechanism of action, other studies have indicated other
possible contributing actions. ADNF-9 has been shown to produce effects
on transcriptional regulation that include an induction of NF-kB
activation in hippocampal neurons (Glazner et al., 2000) and neurite
extension via enhanced cAMP response element-binding protein
phosphorylation in dorsal root ganglia cultures (White et al., 2000).
These studies provide an operational framework to explain the
observation that a short-term exposure to these peptides results in a
long-term protection from toxicity, both in cell culture and in the FAS
model described in the present paper. A transcriptional regulation of
regulators of oxidation/reduction and/or apoptosis mediators may
contribute to the observed long-term protection.
The nervous system is particularly vulnerable to ethanol toxicity at
critical periods during development (Miller, 1995; Olney et al., 2000).
Apoptotic death is produced by the addition of alcohol to neuronal
cultures (Andrews et al., 1999). Recent studies have shown that in
utero exposure to alcohol produced significant increases in apoptosis
in the fetal brain (Ikonomidou et al., 2000). These effects were
particularly evident in the hippocampus, thalamus, and cerebral cortex.
Relevant to these events is previous experiments that demonstrated the
prevention of apoptosis associated with electrical blockade in cerebral
cortical cultures with ADNF, NAP, and ADNF-9 (Brenneman and Gozes,
1996; Brenneman et al., 1998; Bassan et al., 1999). Using a terminal
deoxynucleotidyl transferase assay, ADNF produced inhibition of
apoptosis in cerebral cortical cultures that was evident at femtomolar
concentrations (Gozes et al., 1997). Furthermore, increased hsp60
expression in cortical neurons (Zamostiano et al., 1999) and inhibition
of glutamate-induced calcium levels in hippocampal neurons (Guo et al.,
1999) have been observed after ADNF-9 treatment. From these studies, it
is likely that multiple mechanisms may contribute the protective
properties of ADNF-9 in vivo. Treatment with NAP and ADNF-9 was more
effective when administered in combination. This strongly suggests that
NAP may work through complementary, but different, mechanisms.
Recent studies of NAP and ADNF-9 in other in vivo models provide
additional points of comparison. In apolipoprotein E-deficient mice
(Bassan et al., 1999) and in AF64A-treated animals (Gozes et al.,
2000), NAP was more effective than ADNF-9 in providing long-term
protection against loss of spatial memory. In mice exposed to closed
-head injury, NAP attenuated the increases in tumor necrosis factor-
observed in the brain.
The discovery of protective peptides described herein has its basis in
the neurotrophic and growth-promoting properties of VIP. Although VIP
apparently has fundamental roles in the regulation of embryo/brain
development (Gressens et al., 1993), the peptide itself is quickly
degraded on systemic administration, making it a poor therapeutic
agent. In contrast, several pharmacological properties of NAP and
ADNF-9 form the basis of their unique therapeutic potential: potency at
femtomolar concentrations and apparent stability in vivo, particularly
in the case of NAP. Recovery of significant amounts of radioactive
material comigrating with intact NAP in embryos 60 min after systemic
administration attests to the stability of this peptide. These studies,
in addition to providing a potential important agent for the study of
alcohol-induced damage/death, suggest a promising approach to the
treatment of human neurodegenerative disease that involve oxidative
stress and/or dysregulation of apoptosis.
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Footnotes |
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Accepted for publication January 2, 2001.
Received for publication October 17, 2000.
This study was supported by the Intramural Research Program of the National Institute of Child Health and Human Development and by the U.S.-Israel Binational Science Foundation.
Professor Illana Gozes is the incumbent of the Lily and Avraham Gildor Chair for the Investigation of Growth Factors.
Send reprint requests to: Dr. Catherine Y. Spong, Section on Developmental and Molecular Pharmacology, LDN, National Institute of Child Health and Human Development, National Institutes of Health, Building 49, 5A-38, MSC 4480, 9000 Rockville Pike, Bethesda, MD 20892. E-mail: spongc{at}exchange.nih.gov
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Abbreviations |
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FAS, fetal alcohol syndrome; VIP, vasoactive intestinal peptide; ADNF, activity-dependent neurotrophic factor; ADNF-9, SALLRSIPA; ADNP, activity-dependent neuroprotective protein; NAP, NAPVSIPQ; DPBS, Dulbecco's phosphate-buffered saline; GSH, reduced glutathione; GSSG, oxidized glutathione; ANOVA, analysis of variance.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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studies on cell replication, lipid-peroxidation and glutathione.
Hepatology
18:
648-659[Medline].an in vitro and in vivo immunohistochemical and ultrastructural study.
Alcohol Alcohol
27:
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