Laboratories of Biochemistry, University of Pennsylvania, School of
Veterinary Medicine, Philadelphia, Pennsylvania
Neonatal administration of monosodium glutamate (MSG) can produce
latently expressed defects in drug metabolism and growth hormone
secretion as well as stunted growth and obesity. Instead of secreting
growth hormone in the masculine episodic profile, plasma hormone
levels are generally undetectable in affected adult male rats.
Moreover, male-specific isoforms of cytochrome P450 (P450; e.g.,
CYP2C11 and CYP3A2), whose combined levels comprise the bulk of the
total hepatic P450 in adult male rats, are similarly undetectable in
these animals. Since "signaling elements" in the masculine episodic
growth hormone profile are solely responsible for the elevated
characteristic male-like expression levels of CYP2C11 and CYP3A2,
suppression of the isoforms in the MSG-treated rats appeared to be
caused by the simple absence of the hormone from the circulation.
However, the reported failures of restored physiologic masculine growth
hormone profiles to correct the P450 defects suggested the occurrence
of direct MSG-induced liver damage independent of the well known
hypothalamic lesions produced by the amino acid. Concurrent
administration of dizocilpine maleate (MK-801), a selective and highly
potent noncompetitive N-methyl-D-aspartate receptor antagonist of glutamate, completely prevented the adverse effects of neonatal MSG treatment on P450 expression, growth hormone secretion, and growth parameters, indicating that the amino
acid-induced defects are solely a result of neuronal (i.e.,
hypothalamic) damage produced at the time of MSG exposure. The
irreversibility of the P450 damage is described as resulting from
secondary defects initially induced by the neuronal lesions.
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Introduction |
Neonatal
exposure to the N-methyl-D-aspartate
(NMDA) receptor agonists glutamate and aspartate, excitatory amino acid
neurotransmitters, can induce a syndrome characterized by stunted
growth and obesity. As adults, affected animals exhibit delayed
abnormalities in both growth hormone secretion and cytochrome P450
(P450) expression (Shapiro, 1992
; Agrawal and Shapiro, 1997).
Circulating growth hormone profiles in rats as well as other species
have been shown to be sexually dimorphic (Shapiro et al., 1995). Male
rats secrete growth hormone in episodic bursts (~200 to 300 ng/ml of
plasma) every 3.5 to 4 h. Between the peaks, growth hormone levels
are undetectable. In females, the hormone pulses are more frequent and
irregular and are of lower magnitude than those in males, whereas the
interpulse concentrations of growth hormone are always measurable
(Shapiro et al., 1989; Pampori et al., 1991a). These gender differences
in the circulating growth hormone profiles are responsible for the
dimorphisms in at least a dozen sex-dependent isoforms of P450 observed
in rats and mice (Legraverend et al., 1992; Shapiro et al., 1995).
In the case of CYP2C11, the dominant male-specific isoform comprising
~50% of the total hepatic P450 in the male rat (Morgan et al.,
1985), a female-like pattern of continuous growth hormone secretion
completely blocks expression of the isoform, whereas total growth
hormone depletion from the circulation allows CYP2C11 expression at 20 to 30% of intact male levels (Legraverend et al., 1992; Pampori and
Shapiro, 1996). Whereas normal expression of CYP2C11 requires exposure
to the episodic "on/off" masculine growth hormone profile, the
profile need only be minimal. That is, in contrast to the physiologic
masculine hormone profile, full expression of CYP2C11 requires exposure
to plasma pulses of only 25 ng/ml [or much less (Agrawal and Shapiro,
2000)], at least once, or perhaps less than once every 12 h,
representing <2 to 3% of the masculine physiologic growth hormone
secretory output. Irrespective of the pulse amplitudes, the inductive
"signal" in the masculine profile is a minimum growth
hormone-devoid interpulse of between 100 and 140 min for full CYP2C11
expression (Waxman et al., 1991; Agrawal and Shapiro, 2001). Another
male-specific isoform, CYP3A2 (as well as CYP2A2), is maximally
expressed in the growth hormone-depleted, hypophysectomized rat, is
somewhat suppressed by physiologic pulse amplitudes in the masculine
episodic hormone profile, but completely disappears when exposed to a
continuous pattern of growth hormone secretion representing <3% of
the normal feminine profile (Pampori and Shapiro, 1996).
As adults, male rats neonatally treated with MSG (4 mg/g of body
weight) have neither detectable levels of circulating growth hormone
nor measurable concentrations of hepatic CYP2C11 and CYP3A2 (Shapiro et
al., 1989; Pampori et al., 1991b). Since MSG destroys differentiating
hypothalamic nuclei destined to regulate growth hormone secretion
(Millard et al., 1982; Bloch et al., 1984), it has seemed reasonable to
conclude that the delayed suppression of CYP2C11 and CYP3A2 is due
solely to the permanent absence of growth hormone from the circulation
(Pampori et al., 1991b). However, restoration of physiologic masculine
plasma growth hormone profiles to neonatally MSG-treated, growth
hormone-depleted adult male rats could not restore normal expression
levels of CYP2C11 and CYP3A2; this is in contrast to the complete
effectiveness of the growth hormone treatment when given to
hypophysectomized rats (Shapiro et al., 1993; Waxman et al., 1995). The
abnormal responsiveness of P450 isoforms to growth hormone regulation
may have resulted from MSG-induced defects in hepatic development
independent of the hypothalamic damage produced by the amino acid. To
test this hypothesis, we selectively blocked the neuronal effects of
MSG by simultaneously administering the noncompetitive NMDA receptor blocker dizocilpine maleate [MK-801 (Wong et al., 1988)] and
determined subsequent CYP2C11 and CYP3A2 expression levels and
circulating growth hormone profiles in adulthood.
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Materials and Methods |
Animals.
Animals were housed in the University of
Pennsylvania Laboratory Animal Resources facility under the supervision
of certified laboratory animal medicine veterinarians and were treated
according to a research protocol approved by the University's
Institutional Animal Care and Use Committee. At all times, animals were
housed on hardwood bedding in plastic cages, with water and commercial rat diet supplied ad libitum. The animal quarters were air conditioned (20-23°C) and had a photoperiod of 12 h of light, 12 h of
darkness (lights on at 8:00 AM). After a 2- to 3-week acclimation
period in our facility, the animals were bred by randomly housing two adult female Sprague-Dawley rats [Crl:CD(SD)BR] with an individual adult male of the same strain. On the day of parturition, all litters
were mixed and randomly assigned to the dams at 10 pups per litter,
with a sex ratio of 1:1 or as close to that as possible. Starting
within 24 h after birth, and on alternate days for the first 9 days of life, all the pups in a litter were injected s.c. with either 2 or 4 mg/g of body weight of monosodium L-glutamate (Sigma-Aldrich, St. Louis, MO) or the equivalent NaCl diluent or
1 µg/g of body weight of MK-801 (Sigma/RBI, Natick, MA), or the
equivalent phosphate-buffered saline diluent or both MSG and MK-801 for a total of five injections. In the latter group, pups were
injected on alternate flanks with MK-801 30 min preceding MSG
administration. All pups in a litter received the same treatment. The
pups were weaned at ~24 days of age. At around 25 weeks of age, at
least six male rats in each treatment group were decapitated. Livers
were quickly removed for microsome preparation.
Western Blots.
Hepatic microsomes were isolated as
previously described (Agrawal et al., 1991) and assayed by Western
blotting for the presence of CYP2C11 and CYP3A2 proteins (Pampori et
al., 1995). Microsomal protein (10 µg) was electrophoresed on
0.75-mm-thick SDS-polyacrylamide gels containing 7.5% acrylamide and
electroblotted onto nitrocellulose filters. The blots were probed with
monoclonal anti-rat CYP2C11 (Oxford Biomedical Research Inc., Oxford
MI) and anti-rat CYP3A2 (BD Gentest, Woburn, MA).
The primary antibody was located with horseradish peroxidase conjugated
to anti-mouse or anti-goat IgG and detected with an enhanced
chemiluminescence kit (Amersham Biosciences, Des Plaines, IL).
Quantification of relative P450 levels was done by laser densitometry
of the X-ray films, normalized to a positive control run on every blot.
RNA Analysis.
Total hepatic RNA was isolated by using a
single-step guanidinium thiocyanate method (Chomczynski and Sacchi,
1987). RNA (10 µg) was electrophoresed under formaldehyde-denaturing
conditions on 1% agarose and transferred to GeneScreen nylon membranes
(PerkinElmer Life Sciences, Boston, MA). The Northern blots were probed
and reprobed with 32P-labeled oligonucleotide
probes, using hybridization and high stringency washing conditions as
described previously (Waxman, 1991). The nucleotide sequence of
oligonucleotide probes for CYP2C11 (Waxman, 1991) and CYP3A2 (Ram and
Waxman, 1991) have been reported. The consistency of RNA loading
between samples was confirmed by ethidium bromide staining of 18S and
28S ribosomal RNAs and was verified using an 18S oligonucleotide probe
(Ramsden et al., 1993). The hybridized mRNA signals were quantified by
scanning the autoradiographs and normalized to the 18S rRNA signals in
each lane.
Blood Collection.
Three to four male rats (6-7 months of
age) from vehicle, MK-801, 4 mg of MSG, and 4 mg of MSG plus MK-801
treatment groups were implanted with chronic indwelling right atrial
catheters (Pampori et al., 1991a). Use of our mobile catheterization
apparatus permitted repetitive blood sampling from unrestrained,
unstressed, and completely conscious animals. Blood collections began 5 to 7 days after surgery. During sampling, 25 µl of blood were removed every 15 min for 8 consecutive h. Blood was centrifuged, and 10 µl of
plasma aliquots were stored at 
70°C for future assay. Blood was
collected a second time from each animal 7 to 10 days later following
the same procedure. To maintain extended patency, catheters were
flushed two times per day with heparinized (10 IU/ml) saline.
Growth Hormone.
Eight-hour plasma growth hormone profiles
were determined by using a homologous radioimmunoassay with a
sensitivity of 2 to 3 ng/ml. All values were normalized by subtracting
values obtained from hypophysectomized rats. Procedural details and
statistical validation of the assay have been reported by us elsewhere
(Shapiro et al., 1989; Agrawal et al., 1991).
Sleeping Times.
At 5 to 6 months of age, barbiturate-induced
sleeping times were measured in some of the male rats, representing all
treatment groups, after an i.p. injection of hexobarbital (150 mg/kg of body weight). Recovery from unconsciousness was indicated by the full
restoration of the righting response, defined as the ability of the
animal when placed on its back on a flat surface to snap over on its
paws three times within 15 s (Shapiro et al., 1989).
Statistics.
Data were subjected to analysis of variance, and
differences were determined with t statistics and the
Bonferroni procedure for multiple comparisons.
| |
Results |
Growth.
The effects of neonatal treatment with MSG or MK-801
on body weight gain became apparent only after puberty (Fig.
1, top panel). In agreement with previous
reports (Shapiro et al., 1989; Veneroni et al., 1990), neonatal
exposure to MK-801 alone or 2 mg of MSG alone resulted in a persistent
10 to 15% decrease (P < 0.05 at almost all
postpubertal ages) in body weight, whereas the higher dose of the food
additive (4 mg of MSG) produced a long-lasting ~30% decrease in body
weight (P < 0.01) compared with the vehicle treatment
group.1 Because
MK-801 alone had as similar a depressive effect on body weight gain as
2 mg of MSG alone, it is not surprising that the combined treatment
resulted in no protective effect by the NMDA receptor antagonist on
body weight (Fig. 1, bottom panel). In contrast, concurrent
administration of MK-801 completely prevented the deleterious effects
of the 4-mg MSG dose on subsequent body weight gain.
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Fig. 1.
Body weight gain of male rats treated neonatally with
MSG and/or MK-801. Pups were injected with either 2 or 4 mg of MSG/g of
body weight and/or 1 µg of MK-801/g of body weight or an equivalent
volume of vehicle on days 1, 3, 5, 7, and 9 of life. Results are
presented as the mean of at least 10 rats/data point.
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The effects of neonatal exposure to MSG or MK-801 on tail length, a
measure of skeletal growth, was clearly expressed before or very early
in puberty (Fig. 2, top panel). Neonatal
administration of MK-801 alone or 2 mg of MSG alone produced a similar
<10% decrease (P < 0.05) in tail length, whereas
treatment with the 4-mg dose of MSG resulted in a persistent ~35%
reduction (P < 0.01) in tail length throughout the
4-month study period. Having similar effects when administered
separately, there was expectedly no combined effect (P > 0.05 at almost all ages) of MK-801 and 2 mg of MSG on tail length
(Fig. 2, bottom panel). In contrast, concurrent administration of the
NMDA receptor antagonist dramatically reduced (P < 0.01, although not quite to normal) the permanent effects of the 4-mg
dose of MSG on tail growth.
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Fig. 2.
Tail growth of male rats treated neonatally with MSG
and/or MK-801. Pups were injected with either 2 or 4 mg of MSG/g of
body weight and/or 1 µg of MK-801/g of body weight or an equivalent
volume of vehicle on days 1, 3, 5, 7, and 9 of life. Tail length was
measured from the anus to the tip of the tail. Results are presented as
the mean of at least 10 rats/data point.
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Although tail length is an indicator of skeletal growth, the Lee index
is a measure of obesity (Shapiro et al., 1989). In this regard, whereas
neonatal treatment with MK-801 induced a delayed but small, persistent
decrease in both body weight gain and tail length, the changes were
proportional because there was no evidence of obesity (Table
1). In contrast, MSG administration induced a long-lasting, dose-dependent obesity that was hardly noticeable at the 2-mg dose but was startlingly apparent in the 4-mg-treated male rats. Neonatal administration of MK-801 prevented this MSG-induced obesity. Likewise, concurrent administration of MK-801
with either dose of MSG prevented the subnormal kidney, pituitary, and
seminal vesicle weights observed in adults neonatally exposed to the
amino acid alone (Table 1).
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TABLE 1
Body dimensions, organ weights, and hexobarbital-induced sleeping times
of 6-month-old male rats treated neonatally with MSG and/or MK-801
Pups were injected with either 2 or 4 mg of MSG per gram of body weight
and/or 1 µg of MK-801 per gram of body weight, or an equivalent
volume of vehicle on days 1, 3, 5, 7, and 9 of life.
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Drug Metabolism and Hepatic P450 Levels.
Hexobarbital-induced
sleeping time, a functional measure of drug action, is directly
dependent on but inversely related to hepatic microsomal hexobarbital
hydroxylase activity (Pampori et al., 1991b). We found that neonatal
exposure to either MK-801 alone or 2 mg of MSG alone had no effect on
hexobarbital-induced sleeping time when determined in adulthood (Table
1). In contrast, neonatal administration of the higher 4-mg dose of MSG
resulted in adult sleep times that were twice as long as those observed in control rats. However, concurrent administration of MK-801 completely prevented this effect of 4 mg of MSG.
Prolonged sleeping times are undoubtedly due to a commensurate decline
in hexobarbital hydroxylase activity (Shapiro and Szczotka, 1984;
Pampori et al., 1991b). Since microsomal hexobarbital hydroxylase represents the contributing activities of several isoforms of P450, of
which CYP2C11 and CYP3A2 are dominant (Ryan and Levin, 1990), we
determined the expression levels of these male-specific P450s in our
animals. As previously reported (Shapiro et al., 1989; Pampori and
Shapiro, 2000) neonatal administration of 2 mg of MSG/g of body weight
produces a permanent, albeit small (~20%), over-expression of
CYP2C11 protein that was not corrected by coadministration of MK-801
(Fig. 3). Neonatal treatment with 4 mg of
MSG virtually eliminated CYP2C11 protein levels in treated rats.
Concurrent treatment with MK-801 not only prevented the suppression of
CYP2C11 in male rats treated with 4 mg of MSG, but also induced a
slight but persistent over-expression of the isoform.
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Fig. 3.
Hepatic CYP2C11 and CYP3A2 protein levels in
6-month-old male rats neonatally treated with MSG and/or MK-801. Pups
were injected with either 2 or 4 mg of MSG/g of body weight and/or 1 µg of MK-801/g of body weight or an equivalent volume of vehicle on
days 1, 3, 5, 7, and 9 of life. Each data point is a mean ± S.D.
with n  6 and expressed as a percentage of the
mean value of the vehicle-treated control here designated as 100%.
 , P < 0.05; compared with the vehicle
control.
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Adult levels of CYP3A2, another male-specific isoform, were unaffected
by neonatal exposure to MK-801 or 2 mg of MSG. Like CYP2C11, neonatal
administration nearly eliminated all traces of CYP3A2 protein in adult
liver. Coadministration of the NMDA receptor antagonist was fully
effective in blocking the suppressive effects of 4 mg of MSG on adult
CYP3A2 protein (Fig. 3).
In the absence of substantial effects of the 2-mg dose of MSG on
CYP2C11 and CYP3A2 protein levels, we limited mRNA measurements to rats
exposed to the higher 4-mg dose of MSG. In this regard, mRNA levels
were very much in agreement with protein findings. Whereas neonatal
administration of MK-801 alone had no effect on CYP2C11 mRNA levels at
6 months of age, exposure to 4 mg of MSG nearly eliminated adult
expression of the isoform (Fig. 4). Concurrent administration of MK-801 to the newborns not only prevented the suppressive effects of 4 mg of MSG given neonatally but, like the
protein levels, also caused a small over-expression of the transcript
in adulthood.
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Fig. 4.
Hepatic CYP2C11 and CYP3A2 mRNA levels in 6-month-old
male rats neonatally treated with MSG and/or MK-801. Pups were injected
with 4 mg of MSG/g of body weight and/or 1 µg of MK-801/g of body
weight or an equivalent volume of vehicle on days 1, 3, 5, 7, and 9 of
life. Each data point is a mean ± S.D. with n 6 and expressed as a percentage of the mean value of the
vehicle-treated control here designated as 100%. ,
P < 0.01; compared with the vehicle control.
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Similarly, neonatal treatment with MK-801 alone had no effect on adult
levels of CYP3A2 mRNA (Fig. 4). Whereas adult concentrations of CYP3A2
mRNA were fully suppressed by neonatal treatment with 4 mg of MSG,
coadministration of MK-801 completely blocked the suppressive effects
of the amino acid on expression levels of the transcript.
Growth Hormone Profiles.
Because expression levels of both
CYP2C11 and CYP3A2 are profoundly regulated by sex-dependent signals in
the circulating profiles of growth hormone (Agrawal and Shapiro, 2000,
2001), we examined this relationship in adult rats exposed as neonates to MSG and/or MK-801. The established masculine growth hormone profile
(Shapiro et al., 1989; Pampori et al., 1991a) was found in our adult
vehicle-treated male rats (Fig. 5).
Generally, growth hormone was released in pulses approximately every 3 to 4 h, resulting in short-lived peaks of 200 to 250 ng/ml of
plasma, followed by ~2.5 h of undetectable (<2-3 ng/ml) trough
levels. Neonatal treatment with MK-801 resulted in no alterations in
the masculine profile in adulthood. Although there were basically no
measurable levels of growth hormone in any of the plasma samples
obtained during 8 continuous h of serial blood collections from adult
males treated neonatally with 4 mg of MSG, concurrent administration of
MK-801 completely abolished the deleterious effects of the amino acid, resulting in normal circulating masculine growth hormone profiles in
adulthood (Fig. 5).
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Fig. 5.
Circulating levels of plasma growth hormone in male
rats treated neonatally with MSG and/or MK-801. Pups were injected with
either 4 mg of MSG/g of body weight and/or 1 µg of MK-801/g of body
weight or an equivalent volume of vehicle on days 1, 3, 5, 7, and 9 of
life. Plasma was obtained from 6- to 7-month-old individual,
undisturbed, catheterized rats at 15-min intervals for 8 continuous h.
Similar results were obtained from two to three additional animals in
each treatment group.
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Discussion |
MK-801 is a highly potent and selective noncompetitive NMDA
receptor antagonist (Wong et al., 1988) known to prevent the
neurodegeneration normally induced by cerebral asphyxia (Ford et al.,
1989) and glutamate toxicity (Lehmann and Jonsson, 1992) in neonatal
rats and mice. In the present report, the long-lasting deleterious effects of neonatal exposure to MSG were effectively blocked by concurrent administration of MK-801. That is, neonatal MSG (4 mg/g of
body weight) produced a persistent suppression in body weight gain,
linear growth, and measured organ weights; a notable obesity; a
doubling of hexobarbital-induced sleeping times; and repression of
hepatic CYP2C11 and CYP3A2 expression as well as suppression of plasma
growth hormone secretion to barely detectable levels. All of these
effects were prevented by MK-801, which supports the contention that
the adult suppression of P450s by neonatal MSG can be explained by the
permanent absence of growth hormone from the circulation. However, if
the suppression of male-specific CYP2C11 and CYP3A2 in MSG-treated rats
is simply due to an absence of the masculine plasma growth hormone
profile (and its intrinsic inductive signals), it is unclear why the
renaturalized physiologic profile was far from effective in restoring
male-like levels of the isoforms. In comparison, restoration of the
same masculine growth hormone profile to another model of growth
hormone depletion, the hypophysectomized rat, was completely effective
in restoring male-like levels of CYP2C11 and CYP3A2 (Shapiro et al.,
1993; Waxman et al., 1995).
This apparent inconsistency in the different response of hepatic
CYP2C11 and CYP3A2 to growth hormone replacement in hypophysectomized and MSG-treated rats may be related to the fact that the former experiences a limited, postsurgical period of growth hormone depletion, whereas the neonatally MSG-treated rat lacks growth hormone for its
entire life, including the critical period of differentiation of the
hypothalamic-pituitary-hepatic axis (Gustafsson et al., 1977). This
early growth hormone deficiency may interfere with the development of
the growth hormone receptor and/or the signal transduction mechanism(s)
that normally mediate growth hormone regulation of P450 expression
(Waxman and Frank, 2000), which in turn could result in a permanent
insensitivity of the liver to normal growth hormone secretory profiles.
Alternatively, MSG-treated rats may not be totally devoid of
growth hormone but may actually secrete hormone levels that are too low
for detection by radioimmunoassay. Unlike the hypophysectomized rat,
the MSG-treated rat has a pituitary, albeit containing greatly reduced
levels of growth hormone (Shapiro et al., 1986). Although MSG-induced
lesions in the arcuate nucleus profoundly inhibit secretion of growth
hormone-releasing factor (Millard et al., 1982; Bloch et al., 1984), a
requisite for pituitary growth hormone secretion, the pituitaries of
affected rats are responsive to growth hormone-releasing factor (Dhir
et al., 2002), and it is possible that low, continuous levels of
hormone "leak" from the pituitary. We have found that plasma from
MSG-treated rats consistently displaces a very small amount of
radioactive growth hormone ligand from its specific antibody in our
radioimmunoassay (N. A. Pampori and B. H. Shapiro,
unpublished data), suggesting the possible presence of very low plasma
levels of hormone in these rats. Unfortunately, the displacement is so
small that it extrapolates below the sensitivity of the assay (<3
ng/ml) and cannot be statistically validated. Accordingly, restoration
of a feminine profile of continuous growth hormone secretion at
subdetectable levels, i.e., 1 to 2 ng/ml of plasma, can substantially
suppress expression levels of CYP2C11 and CYP3A2 in male rats (Pampori
and Shapiro, 1999). Moreover, the suppressive effect of these
subdetectable levels of a continuous hormone profile are sufficiently
potent to block the inductive effects of the normal masculine episodic
growth hormone profile on CYP2C11 and CYP3A2 expression (Pampori et
al., 2001). Thus, it is possible that the presence of very low
concentrations of continuously secreted growth hormone in MSG-treated
male rats inhibits the inductive effect of the restored masculine
profile on CYP2C11 and CYP3A2.
In summary, the present findings indicate that the permanent, but
latently expressed, defects in drug metabolism and growth hormone
secretion induced by neonatal administration of MSG are solely a result
of neuronal (i.e., hypothalamic) damage produced through the NMDA
receptor at the time of MSG exposure. The irreversibility of the
damage, however, may be due to secondary defects resulting from
the initial neuronal lesions; i.e., abnormal imprinting of the
developing hypothalamic-pituitary-hepatic axis regulating P450
expression and/or pituitary secretion of continuous, but subdetectable,
growth hormone levels.
Materials used to assay rat growth hormone were obtained through
the National Hormone and Pituitary Program, National Institute of
Diabetes and Digestive and Kidney Diseases, and Dr. A. F. Parlow. We thank Dr. Douglas H. Smith for advice on MK-801.
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Abstract
Introduction
-hydroxylase cytochrome P-450 apoprotein in the rat.
J Biol Chem
260:
11895-11898
